Microstructural analysis on the testicular cyst of the wolf spider by 3D volume rendering

Spermatogenesis has long been a major research area in understanding the development of living organisms. In vertebrates, sperm is produced along the wall of the seminiferous tubules, leaving spermatogonia in the outermost layer, which undergo cell division and differentiation. However, sperm in many invertebrates is developed in a testicular cyst, which contains germ cells at the same developmental stages. On the contrary, in spiders, it is very difficult to count the exact number of cells in a cyst, since each spermatid gets transformed to a round sperm ball within the cyst through the flagellar coiling process. Therefore, in this study, we applied a 3D rendering technique to analyze the exact number of germ cells per cyst in spiders. For image processing and reconstruction, serial section images were scanned and reconstructed into 3D images. Upon successful 3‐dimensional reconstruction of testicular cysts, the exact number of germ cells produced from a single cyst appeared to be 64, 2⁶ which indicates that a spider spermatogonium undergoes 6 cell divisions to produce spermatozoa.     

Structural analysis of theridiid spider's testicular cyst using 3D reconstruction rendering

A three dimensional reconstruction technique was used for the analysis of a theridiid spider's (Achaearanea tepidariorum) testicular cyst. Although microscopic techniques have greatly improved, most of the information gathered is still based on two‐dimensional images. Particularly in spiders, it is very difficult to count the exact number of sperm in a single cyst, since their spermatogenetic processes takes place within the spherical cysts through the flagellar coiling process. Since morphological features of spider sperm provide detailed information on the whole spermatogenetic processes, we analyzed the exact number of germ cells per cyst in A. tepidariorum through a three‐dimensional image reconstruction technique. For image processing, serially sectioned histological images were scanned using a light microscope and 3D rendering images were reconstructed from these sections. Based on the three dimensional image analysis of the testicular cyst, the number of secondary spermatocytes per cyst was calculated to be 32 (2⁵). Therefore the total number of sperm produced from a single cyst can be calculated as 64 (2⁶), which indicates that a single spermatogonium undergoes four mitotic divisions and an additional two meiotic divisions to produce mature spermatozoa.     

Fine structural reconstruction on the testicular cyst of the furrow orb weaver,Larinioides cornutus by 3D volume rendering

Morphological study on spermatids and spermatozoa have long been performed regarding various changes of cell organelles during spermiogenesis as a potential phylogenetic inference. Based on the fact that the number of germ cells per cyst increases according to a geometric series, knowing the exact number of germ cells in a certain stage may lead to the total number of sperms produced per cyst. In spiders, however, the entire process takes place in a cyst represented by a spermatogonium, producing sperms in spherical shape. It is very difficult to count the exact number of germ cells produced per cyst through a 2D image analysis. Therefore, we applied a 3D image of testicular cyst of an orb-weaving spider to visualize the exact number of germ cells produced from a cyst. In this study, 2D images obtained from serially sectioned micrographs were scanned precisely and reconstructed using a 3D-rendering technique. Finally, this research reveals that the exact number of spermatozoa produced each cyst in Larinioides cornutus appeared to be 128 (2⁷), which indicates that a single spermatogonium undergoes five mitotic divisions and two maturing divisions (meiosis) to produce final spermatozoa.     

Evidence for asynchronous mitotic cell divisions in secondary spermatogonia of Drosophila

Examination of germ cell numbers within premeiotic as well as postmeiotic cysts of various Drosophila species gave evidence against any strict synchrony of mitotic cell division in secondary spermatogonia. The evidence was based on numbers of germ cells in primary spermatocyte cysts and spermatid bundles. Each species examined had its own distribution of primary spermatocyte cyst types in pupal testes, and the most common cyst type did not necessarily contain 2, 4, 8, 16 or (2) ⁿ germ cells which implies asynchrony of the previous spermatogonial divisions. Similar but not exactly the same distributions of germ cells were found in adult spermatid bundles, if allowances were made for a 4-fold increase in germ cell number during meiosis. This observation gives support to the operation of an age-dependent factor which controls germ cell numbers within cysts [1], The data thus suggest that the commonly accepted concept of a (2) ⁿ increase of spermatogonia via synchronous mitotic divisions is not true for the species of Drosophila studied.     

Cytology of lepidoptera. III. Giant cysts: A morphological trait of apyrene spermatogenesis in an Ephestia kuehniella strain

A comparative investigation of testicular eupyrene cysts (in larvae) and apyrene cysts (in pupae) of Ephestia kuehniella laboratory strains was conducted using light and electron microscopy. Eupyrene cysts in the first meiotic division contained 64 spermatocytes, which showed only moderate asynchrony. In one of the strains, a wild-type strain, L, normal-sized cysts occurred together with abnormally large cysts. These are called giant cysts in this article. One of the premeiotic cysts, early giant cysts, studied in detail, contained approximately a fourfold number of cells compared with the number in a eupyrene cyst of the same stage. In cysts harboring spermatocytes and spermatids, late giant cysts, cell differentiation was highly asynchronous. Failure in one of two control mechanisms in early cyst development may have caused the appearance of the cysts. Control of cell division might have been sloppy in apyrene spermatogonia. Hence, the spermatogonia within the cyst could have passed through additional division cycles. Alternatively, the giant cysts may have originated from more than one predefinitive gonial cell enclosed in a common envelope of sheath cells. As a third possibility, giant cysts could have arisen by fusion of normal cysts at a later stage. In either case, this is evidence that separation of eupyrene and apyrene pathways is earlier than was previously expected. In two other Ephestia strains, apyrene sperm development proceeded without formation of giant cysts. One was a mutant strain, a, and the other one was a recently established wild-type strain, Sbr. Apyrene sperm development is considered an example of degenerate evolution in which enhanced variability between species and even between populations of one species is a common phenomenon.     

Evidence against a (2)n synchronous increase of spermatogonia to produce spermatocytes in drosophila hydei

The numbers of primary spermatocytes within cysts as well as numbers of postmeiotic spermatids in bundles in Drosophila hydei were determined. Within the contents of a single testis the cysts of primary spermatocytes are found to contain 5–11 germ cells. Furthermore, the number of spermatocytes per cyst is age-dependent, in that pupae have a mean of 8.1 cells whereas fertile adult males have a mean of 7.1 cells. Counts of spermatids in section of testes add further support to the view that the primary spermatocytes, from which the spermatids originated, were not formed in a strict geometric progression.     

Intercellular bridges and factors determining their patterns in the grasshopper testis

Intercellular bridges joining cells contained in cysts of Chortophaga viridifasciata testes were studied with light and electron microscopy. Preparations consisted of expressed whole cells (living, or fixed and stained) as well as sections. The secondary spermatogonia of each cyst are joined centrally by persisting fused interzonal bodies (fusomes) of incompletely cleaved cells. Shifts in cell orientation during anaphase are apparently responsible for central as opposed to chain linkage of cells. In the primary spermatocytes, the central fusome is replaced by a chain linkage, apparently resulting from the breakdown of the fusome into its original interzonal body components. Intercellular bridges are also present in spermatids, but there is no evidence to indicate the time of their formation (in the immediately preceding meiotic divisions or in the secondary spermatogonial divisions). The function of the compact centrally situated fusome in the secondary spermatogonial cyst is discussed as it relates to synchrony, number of cell divisions, spermatodesm formation, and fertility.     

Sperm development in the teleost Oryzias latipes

Summary In Oryzias latipes the processes of spermatogenesis and spermiogenesis occur within testicular or germinal cysts which are delimited by a single layer of lobule boundary cells. These cells, in addition to comprising the structural component of the cyst wall, ingest residual bodies cast off by developing spermatids. Therefore, they are deemed to be the homologue of mammalian Sertoli cells. The germ cells within a cyst develop synchronously owing to the presence of intercellular bridges connecting adjacent cells. Since bridges also connect spermatogonia, it seems probable that all of the germ cells within a cyst may form a single syncytium and do not exist as individual cells until the completion of spermiogenesis when the residual bodies are cast off. Significant differences between spermiogenesis in O. latipes and in the related poeciliid teleosts are discussed.     

Cytoplasmic bridges,intercellular junctions,and individualization of germ cells during spermatogenesis in Dermatobia hominis (Diptera: Cuterebridae)

During mitotic and meiotic divisions in Dermatobia hominis spermatogenesis, the germ cells stay interlinked by cytoplasmic bridges as a result of incomplete cytokinesis. By the end of each division, cytoplasmic bridges flow to the center of the cyst, forming a complex, called the fusoma. During meiotic prophase I, spermatocytes I present desmosome-like junctions and meiotic cytoplasmic bridges. At the beginning of spermiogenesis, the fusoma moves to the future caudal end of the cyst, and at this time the early spermatids are linked by desmosome-like junctions. Throughout spermiogenesis, new and sometimes broad cytoplasmic bridges are formed among spermatids at times making them share cytoplasm. In this case the individualization of cells is assured by the presence of smooth cisternae that outline their structures. The more differentiated spermatids have in addition to narrow cytoplasmic bridges, plasmic membranes junctions. By the end of spermiogenesis, the excess cytoplasmic mass is eliminated leading to spermatid individualization. Desmosome-like junctions of spermatocytes I and early spermatids appear during the fusoma readjustment and segregations; on the other hand, plasmic membrane junctions appear in differentiating spermatids and are eliminated along with the cytoplasmic excess. These circumstances suggest that belt desmosome-like and plasmic membrane junctions are involved in the maintenance of the relative positions of male germ cells in D. hominis while they are inside the cysts. © 1996 Wiley-Liss, Inc.     

Biosynthesis and localization of lactate dehydrogenase X in pachytene spermatocytes and spermatids of mouse testes

The presence and biosynthesis of the testis-specific isozyme of lactate dehydrogenase (LDH-X) in cells at various stages of spermatogenesis have been examined. Enrichment of testicular cells in various stages of spermatogenesis has been achieved by two methods: (1) cell separation by velocity sedimentation in the Elutriator rotor and (2) γ irradiation of testes to eliminate specific classes of testicular cells. Separation of cells from immature mice indicated that cells prior to the midpachytene stage contain no LDH-X. Measurement of LDH-X levels in cells separated from adult mice and in testicular homogenates prepared at various times after irradiation indicated that the highest level of LDH-X per cell (normalized for DNA content) was in spermatids. Synthesis of LDH-X was determined, after in vivo injection of [³H]valine, by measurement of the radioactivity in LDH-X precipitated with specific antiserum. After irradiation, the rate of LDH-X synthesis remained constant, despite the loss of early primary spermatocytes. In separated cells, the rate of LDH-X synthesis was highest in late pachytene spermatocytes, lower in round spermatids, and even lower, but still significant, in elongated spermatids. Therefore, the synthesis of LDH-X begins at a specific point during spermatogenesis, the midpachytene stage of spermatocyte development, and continues throughout spermatid differentiation.     

Gap junctions between germ and somatic cells in the testis of the moth,Anagasta küehniella (Insecta: Lepidoptera)

Summary Heterocellular gap junctions were demonstrated in germ cysts of the moth Anagasta küehniella (Lepidoptera). They conjoin peripheral germ cells of a cyst and cells of their envelope. Their morphology differs according to the developmental stage of the germ cell involved. While gap junctional profiles are flat in cysts of gonia, in cysts of early spermatocytes they appear as button-like structures, the germ cell indenting the corresponding cyst cell. In cysts of late spermatocytes and of young spermatids, they are very numerous and often located at the extremity of conical protrusions of the germ cell. On the germ cell side, cytoplasmic microfilaments are associated with the junctional differentiation. Gap junctions are observed as being pinched off from the surface of the spermatids and, correspondingly, gap vesicles are found in the cyst cells. This, together with the fact that gap junctions are not found at later stages of development, suggests that internalization of the gap junctions might take place before elongation of the spermatids. The potential importance of these germsomatic cell gap junctions is evaluated in light of recent physiological findings obtained by other authors on the oocyte-cumulus system and also in relation with some particularities in the development of the male germ cells in Lepidoptera.     

Intratesticular Epidermoid Cyst Masquerading as Testicular Torsion

Epidermoid cysts are benign tumors that comprise approximately 1% of all testicular masses. They usually present as painless masses that can be identified on scrotal ultrasound as well-demarcated intratesticular lesions with mixed echogenicity. This case report describes a rare presentation of an extremely large intratesticular epidermoid cyst with clinical and radiologic findings more consistent with testicular torsion. The sizeable cyst obliterated the surrounding testicular parenchyma, causing it to appear on scrotal Doppler ultrasound as a testicle devoid of blood flow. This obliteration also resulted in failure to identify a testicular mass on physical examination or imaging. The current literature contains previous reports of extratesticular epidermoid cysts presenting as torsion; however, this is the first report of an intratesticular epidermoid cyst presenting in this manner. Though smaller cysts may be managed effectively with testicular-sparing surgery, optimal management of a cyst this size requires orchiectomy.Key words: Epidermoid cyst, Testicular torsion, Acute testicular pain, Intratesticular, Doppler ultrasoundIntratesticular epidermoid cysts are relatively rare benign testicular masses that comprise approximately 1% of all testicular tumors.^1–6 An epidermoid cyst typically presents as a painless testicular mass and consequently often mimics the presentation of a malignant testicular neoplasm. This case review details the case of a patient with an unusual presentation of an intratesticular epidermoid cyst—one of acute testicular torsion. To our knowledge, this is the first case of its kind to be reported in the literature.     

Evolution and development of polarized germ cell cysts: new insights from a polychaete worm, Ophryotrocha labronica

Polarized oogenic cysts are clonal syncytia of germ cells in which some of the sister cells (cystocytes) differentiate not as oocytes, but instead as nurse cells: polyploid cells that support oocyte development. The intricate machinery required to establish and maintain divergent cell fates within a syncytium, and the importance of associated oocyte patterning for subsequent embryonic development, have made polarized cysts valuable subjects of study in developmental and cell biology. Nurse cell/oocyte specification is best understood in insects, particularly Drosophila melanogaster. However, polarized cysts have evolved independently in several other animal phyla. We describe the differentiation of female cystocytes in an annelid worm, the polychaete Ophryotrocha labronica. These worms are remarkable for their elegantly simple cysts, which comprise a single oocyte and nurse cell, making them an appealing complement to insects as subjects of study. To elucidate the process of cystocyte differentiation in O. labronica, we have constructed digital 3D models from electron micrographs of serially sectioned ovarian tissue. These models show that 2-cell cysts arise by fragmentation of larger “parental” cysts, rather than as independent units. The parental cysts vary in size and organization, are produced by asynchronous, indeterminate mitotic divisions of progenitor cystoblasts, and lack fusome-like organizing organelles. All of these characteristics represent key cytological differences from “typical” cyst development in insects like D. melanogaster. In light of such differences and the plasticity of female cyst structure among other animals, we suggest that it is time to reassess common views on the conservation of oogenic cysts and the importance of cysts in animal oogenesis generally.     

Impaired spermatogenesis in the Japanese eel, Anguilla japonica: Possibility of the existence of factors that regulate entry of germ cells into meiosis

In the cultivated male Japanese eel, spermatogonia are the only germ cells present in the testis. Weekly injections of human chorionic gonadotropin (HCG) can induce complete spermatogenesis from proliferation of spermatogonia to spermiogenesis. In some cases, however, HCG injection fails to induce complete spermatogenesis. Testicular morphological observations revealed that HCG-injected eels could be classified into three types based on their testicular conditions. Type 1 eels had a well-developed testis and the milt could be acquired by hand-stripping. In type 2 eels, spermatogenesis was also induced by HCG injection, but testicular size was remarkably smaller than that of type 1 eels, and the milt could not be hand-stripped. At the end of the experiment, type 2 fish had only spermatogonia and a small amount of spermatozoa, but no spermatocytes or spermatids, in their testis. Type 3 eels had thready testis, which did not develop any germ cells during the experimental period. These results suggest that, despite elevations of plasma 11–ketotestosterone levels, HCG injections were not successful in inducing the completion of spermatogenesis in type 2 and type 3 eels. In most spermatogonia of type 2 eels, meiosis was not induced by HCG injections. Furthermore, only few mitotic divisions had occurred as evidenced by the presence of 2³ to 2⁶ late type B spermatogonia in most cysts. This suggests that spermatogonial stem cells undergo four or five, and occasionally six, mitotic divisions before the interruption of spermatogenesis in type 2 eels. It is proposed that those numbers of mitotic divisions are related to a mediator that regulates entry of spermatogonia of the Japanese eel into meiosis.     

Increased germ cell degeneration and reduced germ cell:Sertoli cell ratio in stallions with low sperm production

To determine the relationship between germ cell degeneration or germ cell:Sertoli cell ratio and daily sperm production, testes were obtained during the months of May to July (breeding season) and November to January (nonbreeding season) from adult (4 to 20-yr-old) stallions with either high (n = 15) or low (n = 15) sperm production. Serum was assayed for concentrations of LH, FSH and testosterone. Testes were assayed for testosterone content and for the number of elongated spermatids, after which parenchymal samples were prepared for histologic assessment. Using morphometric procedures, the types and numbers of spermatogonia, germ cells and Sertoli cells were determined. High sperm producing stallions had greater serum testosterone concentration, total intratesticular testosterone content, testicular parenchymal weight, seminiferous epithelial height, diameter of seminiferous tubules, numbers of A and B spermatogonia per testis, number of Sertoli cells per testis, and number of B spermatogonia, late primary spermatocytes, round spermatids and elongated spermatids per Sertoli cell than low sperm producing stallions (P < 0.05). The number of germ cells (total number of all spermatocytes and spermatids in Stage VIII tubules) accommodated by Sertoli cells was reduced in low sperm producing stallions (18.6 +/- 1.3 germ cells/Sertoli cell) compared with that of high sperm producing stallions (25.4 +/- 1.3 germ cells/Sertoli cell; P < 0.001). The conversion from (yield between) early to late primary spermatocytes and round to elongated spermatids was less efficient for the low sperm producing stallions (P < 0.05). Increased germ cell degeneration during early meiosis and spermiogenesis and reduced germ cell:Sertoli cell ratio was associated with low daily sperm production. These findings can be explained either by a compromised ability of the Sertoli cells to support germ cell division and/or maturation or the presence of defects in germ cells that predisposed them to degeneration.     

Ultrastructural Study of Spermatogenesis in Gamasid Mites (Acari,Gamasida)

Structural alterations accompanying the development of germ cells in representatives of two families of gamasid mites: Pergamasidae (Pergumasus barbarus. P. truatellus) and Parasitidae (Porrhostaspis lunulata) were examined by scanning electron microscopy. Spermatogenesis occurs in cysts containing two (P. lunuluta) or four spermatogonia (P. barbarus and P. truatellus) prior to meiosis. later arranged in a tetrahedral pattern. Meiosis produces 8 and 16 spermatids per cyst, respectively. Spermatids are located radially and elongate synchronously. Dimples appear at their acrosomal ends, soon replaced by peg-like structures surrounded by superficial ridges. The number of ridges which correspond to longitudinal ribbons can vary between and within species as well as between spermatids occupying one cyst. Our observations suggest some unknown mechanism which allows the spermatozoa to enter vasa deferentia in a unidirectional orientation.     

Impact of Zooplankton Grazing on the Excystation, Viability, and Infectivity of the Protozoan Pathogens Cryptosporidium parvum and Giardia lamblia

Very little is known about the ability of the zooplankton grazer Daphnia pulicaria to reduce populations of Giardia lamblia cysts and Cryptosporidium parvum oocysts in surface waters. The potential for D. pulicaria to act as a biological filter of C. parvum and G. lamblia was tested under three grazing pressures (one, two, or four D. pulicaria grazers per 66 ml). (Oo)cysts (1 × 10⁴ per 66 ml) were added to each grazing bottle along with the algal food Selenastrum capricornutum (6.6 × 10⁴ cells per 66 ml) to stimulate normal grazing. Bottles were rotated (2 rpm) to prevent settling of (oo)cysts and algae for 24 h (a light:dark cycle of 16 h:8 h) at 20°C. The impact of D. pulicaria grazing on (oo)cysts was assessed by (i) (oo)cyst clearance rates, (ii) (oo)cyst viability, (iii) (oo)cyst excystation, and (iv) oocyst infectivity in cell culture. Two D. pulicaria grazers significantly decreased the total number of C. parvum oocysts by 52% and G. lamblia cysts by 44%. Furthermore, two D. pulicaria grazers significantly decreased C. parvum excystation and infectivity by 5% and 87%, respectively. Two D. pulicaria grazers significantly decreased the viability of G. lamblia cysts by 52%, but analysis of G. lamblia excystation was confounded by observed mechanical disruption of the cysts after grazing. No mechanical disruption of the C. parvum oocysts was observed, presumably due to their smaller size. The data provide strong evidence that zooplankton grazers have the potential to substantially decrease the population of infectious C. parvum and G. lamblia in freshwater ecosystems.     

Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: Specific activities and rates of synthesis

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [³H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.     

The distribution and proliferation of the intracellular bacteria Wolbachia during spermatogenesis in Drosophila.

Wolbachia is a cytoplasmically inherited alpha-proteobacterium found in a wide range of host arthropod and nematode taxa. Wolbachia infection in Drosophila is closely associated with the expression of a unique form of post-fertilization lethality termed cytoplasmic incompatibility (CI). This form of incompatibility is only expressed by infected males suggesting that Wolbachia exerts its effect during spermatogenesis. The growth and distribution of Wolbachia throughout sperm development in individual spermatocysts and elongating sperm bundles is described. Wolbachia growth within a developing cyst seems to begin during the pre-meiotic spermatocyte growth phase with the majority of bacteria accumulating during cyst elongation. Wolbachia are predominantly localized in the proximal end of the immature cyst, opposite the spermatid nuclei, and throughout development there appears little movement of Wolbachia between spermatids via the connecting cytoplasmic bridges. The overall number of new cysts infected as well as the number of spermatids/cysts infected seems to decrease with age and corresponds to the previously documented drop in CI with age. In contrast, in one CI expressing line of Drosophila melanogaster, fewer cysts are infected and a much greater degree of variation in numbers is observed between spermatids. Furthermore, the initiation and extent of the fastest period of Wolbachia growth in the D. melanogaster strain lags behind that of Drosophila simulans. The possible implications on the as yet unexplained mechanism of CI are discussed.     

Beziehungen zwischen Vermehrungswachstum und Differenzierung von männlichen Keimzellen von Drosophila melanogaster

Summary A count-chamber like methodes enables us to estimate the number of cysts corresponding to each developmental stage of the spermatogenesis.The multiplication of the germ cells depends upon the physiological conditions of the host: it is stopped in isolated larval abdomina, and is low in larvae of a crowded culture where pupation is delayed. In implants of 48 h old testis in. virgin adult females the multiplication rate is lower than in males and in mated females; it is as high as in situ in castrated females or in pupal hosts.The time required for the oldest cysts with spermatocytes to enter the meiotic divisions is with one exception for all these experiments the same as in situ; only in pupal hosts it will be accelerated. The subsequent spermatid differentiation in sperm also takes the same time as in situ. Thereby the premeiotic growth and postmeiotic differentiation is as efficient in all tested milieus as in situ, even when the hosts have not attained or already bypassed the metamorphosis. This autonomy is also valid for germ cells under insufficient multiplying conditions, provided that the spermatocytes have reached a certain stage in the premeiotic growth.When the first cysts reach the postmeiotic stages the further multiplication of cysts is stopped. This regulative mechanism is attributed to a specific inhibition operated by the spermatids.The autonomy of the spermatocyte differentiation and the unified behaviour of all the cells within a cyst are discussed.

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Herrn Prof. Dr. E.Hadorn bin ich zu Dank verpflichtet für die kritische Diskussion dieser Arbeit, Herrn Prof. Dr. H.Burla für die Unterstützung in statistischen Prägen, Herrn Dr. R.Nöthiger und Herrn W.Gehring für die Hilfe bei der Abfassung des deutschen Textes.