Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 241-258). Available also in a digital version from Dissertation Abstracts.

Testicular maturation indices (TMI) were determined for wild-caught males of Poecilia mexicana by quantitative analysis of the frequency of ongoing stages of spermatogenesis and spermatid differentiation in sections of testes stained with the Feulgen reaction for DNA. In nature, males maintain essentially constant levels of sperm production throughout the year and show no significant variations in mean TMI values associated with season, microhabitat, standard body length, or rank in male dominance hierarchies. Winter males or males isolated from females in the laboratory show accumulation of mature spermatophores. These findings suggest that fluctuations in the microstructure of wild populations of Poecilia from northeastern Mexico may be due primarily to differences in the temporal scheduling of female reproductive cycles and not to limitations imposed by sperm availability or male reproductive competence. The data are discussed in terms of competitive interactions within unisexual-bisexual breeding complexes involving the Amazon molly P. formosa, its related triploid hybrids, and the bisexual species, P. mexicana.

There have been leaps in both fields of epigenetics and reproductive technology. This has culminated in the production of bi-maternal mouse offspring through a few studies utilizing direct gene mutations as functional-imprints. While these genetic interventions result in positive results, it has yet to be described, in full, what mechanisms underlie the epigenetic barriers to human gynogenesis. Between maternal and paternal imprints, differences in methylation patterns of CpG island promoters, non-coding regions, microsatellites, transposons, and histones result in differences in higher order chromatin structure, which ultimately impacts expression of certain genes. While the necessary components of a minimal paternal epigenetic program are described, programming this imprint onto m2, a hypothetical, experimentally-produced maternal genome with a paternal epigenome is still not elucidated. Sequential timing of imprint acquisition and maintenance and the numerous regulatory factors associated with them illuminate how difficult potential assisted reproductive epigenetic interventions will be. Other processes like histone-protamine exchange which also plays a crucial factor in structural regulation of imprints, as well as signaling through and after fertilization, pose logistical problems to creating a gynogenote through epigenetic means. Lastly, ethics surrounding the introduction of dangerous mutations and epialleles into the human population add another wall of caution and hesitance in exploring human gynogenesis as a reproductive technology.

Seven families of XX and YY homozygous Oreochromis niloticus were produced by mitotic gynogenesis from XY neofemales and their genetic status was verified by multilocus DNA fingerprinting and progeny testing. Two of these gynogenetic families and their corresponding diploid controls were used with 64 AFLP primer combinations in different levels of screening (XX/YY grand pool; XX/YY family pool; XX/YY gynogenetics and XX/XY control individuals) to search for sex-linked or sex-specific markers. Grand pool screening did not reveal any sex-linked markers. Subsequent family pool and individual level screening identified four sex-linked AFLP markers from two primer combinations, three Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420). Two of these (OniX420, OniY425) were shown to be allelic. Single locus PCR markers were developed for all of those markers. Linkage analysis of these markers and the sex locus within the source families revealed tight linkage, with estimated map distances of 13cM, 17cM and 20cM for OniY382, OniY227 and OniX420/OniY425 respectively. However, these sex-linked AFLP markers failed to consistently identify sex in unrelated individuals. To develop an effective system for parentage analysis in normal and gynogenetic progeny, AFLPs and multiplexed polymorphic microsatellite loci were investigated. Both were found to be effective, but microsatellites were more appropriate since they are codominant and some loci showed high gene-centromere recombination rates, suitable for discriminating meiotic from mitotic gynogenetics, while AFLPs are dominant markers. Spontaneous diploidization of the maternal chromosome set (SDM) was observed in gynogenetic progeny of one XY neofemale. Maternal inheritance and ploidy status were verified by multilocus DNA fingerprinting and chromosome karyotyping. Close genetic linkage between the red gene and an autosomal sex-reversal gene(s) in gynogenetic progeny and influences of autosomal sex-reversal gene(s) producing males in a fully inbred XX clonal line were previously reported in O. niloticus. To test if the same autosomal sex-reversal locus was responsible in both cases, a series of test crosses was carried out involving XX clonal neomale(s) and homozygous red females. The results indicated the involvement of more than one autosomal sex-reversal locus, one of which is linked to red body colour.

The goal of this thesis was to assess colour inheritance in Showa tricolour variety of koi carp (black basis, white and red signs). Fish of the same colouration were pair-mated. Colouration of fingerlings was assessed in one day age. Colouration was observed in two forms, like melanin pigmentation and non-melanin pigmentation (black- pigmented and gold -pigmented fingerlings). In the tests, gynogenesis was employed, transmissing only mother`s genome to offspring. Gynogenesis was practiced in RIFCH USB in Vodnany. Gynogenetic offspring was further photographically documented to assess colouration of fingerlings and relationship between both variants of colouration. Fry from propagation was bred in ponds to the size stage of advanced fry (Kr; 90-day old, size 5- 6 cm). In this size, fingerlings were caught from breeding ponds, counted and further documented to check phenotype segregation.

Les lignées clonales sont un outil puissant pour une meilleure caractérisation génétique des organismes utilisés en recherche. En utilisant les techniques de reproduction uniparentale, de telles lignées peuvent être développées chez les poissons en seulement deux générations. L'androgenèse avait déjà été réalisée chez plusieurs espèces d'eau douce et a été tentée chez le loup, une espèce d'intérêt économique et scientifique. Le faible nombre d'individus contenant effectivement uniquement le patrimoine génétique du père, après irradiation des œufs aux UV, a soulevé des questions quant à la présence de composés photoprotecteurs dans les œufs pélagiques. Les acides aminés de type mycosporines et le gadusol ont été recensés dans de nombreux organismes marins et d'eau douce, mais leur présence n'a pu être reliée au comportement flottant ou coulant des œufs. Tandis que le gadusol apparaissait en plus grande quantité dans les œufs marins flottants, ce résultat était beaucoup moins clair en eau douce et le type de composés photoprotecteurs semble lié à la phylogénie. D'autres études sur les mécanismes de photoréparation de l'ADN pourraient éclairer les résultats obtenus dans les expériences d'androgenèse. La gynogenèse avait déjà été reportée chez le loup mais le grand nombre d'individus méiotiques contaminant les descendances homozygotes produites a conduit à valider des marqueurs microsatellites efficaces à distinguer les individus mitotiques. De plus, un retard de développement des gonades a été observé, augmentant les difficultés à obtenir les lignées clonales. La grande variabilité observée entre les individus quant au succès des reproductions uniparentales a montré que la caractérisation et la qualité des gamètes sont un préalable indispensable. / Clonal lines are a powerful scientific tool for improved genetic characterization of organisms used in research. Inbred fish lines can be produced in only two generations using uniparental reproduction techniques. Androgenesis, achieved with variable success in several freshwater species, has been attempted in the European sea bass (Dicentrarchus labrax L), a marine fish of commercial and scientific interest. The low yields of progenies inheriting only the paternal genome after UV-irradiation of eggs led to considerations on the occurrence of UV screening compounds in pelagic eggs. Mycosporine-like amino acids and gadusol were found in many marine and freshwater organisms, but their occurrence in fish eggs was not clearly related to a behavioral pattern and while gadusol appeared in higher proportions in pelagic marine eggs compared to benthic species, this statement did not apply in freshwater, and moreover the kind of compounds was related to phylogeny. Further studies on DNA photorepair could enlighten hypotheses to understand the mechanisms underlying the disparate results obtained in inducing androgenesis in different fish species. Gynogenesis was reported successful to produce clonal founders in the sea bass, but high numbers of meiotic individuals contaminating fully homozygous progenies highlighted the need for efficient DNA markers to distinguish mitotic gynogenetic individuals. Furthermore, gonad development was highly delayed in gynogenetic progenies enhancing the difficulties to produce clonal lines. A high variability between individuals in the success of uniparental reproduction brought out gamete characterization and quality as a prerequisite.

There are four members involved in the breeding complexes of poeciliid fishes found in the freshwaters of northeastern Mexico: males and females of a bisexual species, and diploid and triploid unisexuals. Both unisexuals reproduce by gynogenesis, i.e., an asexual type of reproduction where the sperm triggers egg development but the male genome is excluded to produce clonal offspring. The three types of females are closely related, which suggests that they are potential competitors since all three require the service of the same males. The potential for competition is compounded by a highly skewed sex ratio in favor of females. On the average the unisexuals comprise about 30% of the Poecilia females. This high frequency coupled with a close genetic relatedness to their bisexual hosts, raises the question of how the unisexuals are maintained in nature. Other investigators who work with bisexual/unisexual complexes in the related genus, Poeciliopsis, have postulated that male dominance hierarchies are responsible for restricting the access of subordinate males to their conspecific females. Consequently, these subordinate males mate with unisexual females. The current report tests whether or not this hypothesis applies to bisexual/unisexual complexes of Poecilia. We have found that linear dominance hierarchies appear to function in the defense of home ranges and do not restrict access of males to females. Dominant males exhibit less mating activity than subordinate males towards females. Previous reports showed that males are reproductively competent throughout the year, whereas females show striking asynchrony in their reproductive readiness. Such asynchrony limits the proportion of receptive females at any one time. Consequently, there are more males ready to mate than there are females receptive to their mating attempts. This may lead to mating frenzies. We postulate that these indiscriminate matings maintain the fertility of both unisexuals. When the relative reproductive outputs of adult females are compared, both unisexuals appear as fit as their bisexual congeners.

Several species‐specific proteins have been identified by polyacrylamide gel electrophoresis (PAGE) of skeletal muscle extracts from the diploid gynogen, Poecilia formosa, its related triploid unisexuals, and their sympatric, bisexual species, P. mexicana and P. latipinna. These water‐soluble, low molecular weight proteins (7,000‐13,000) comigrate with a fraction of purified rabbit parvalbumin on nondenaturing gels and show staining properties similar to rabbit parvalbumins. The electrophoretic patterns of these muscle proteins provide a set of distinctive phenotypic markers for each of the host species involved in naturally occurring breeding complexes with P. mexicana × P. latipinna show no evidence of sexual dimorphisms. Furthermore, the hybrid phenotypes are those that would be predicted from appropriate combinations of parental alleles at three gene loci. The patterns found by PAGE for several generations of pedigreed stocks of P. formosa show strictly matroclinous inheritance of a characteristic muscle protein phenotype and coupled with the electrophoretic patterns of several enzymic proteins reflect the probable hybrid origin of this diploid unisexual. Finally, paternal contributions by P. mexicana to the hybrid genome of triploid unisexuals are clearly demonstrated by comparative analyses of muscle protein phenotypes for P. formosa and its contemporary host species. Our identification of distinctive phenotypic markers in the muscle proteins of several poeciliid species involved in unisexual‐bisexual breeding complexes provides an important new tool for further studies on the adaptive significance of unisexuality, hybridization, and fixed heterozygosity in the evolutionary biology of these fishes.

No description available.

Highly profitable black caviar market and the depletion of wild sturgeon stocks warrant improvements in sturgeon aquaculture. Therefore, chromosomal manipulations, particularly gynogenesis, are focused on for increasing the ratio of females over males in progeny. The present study focused on optimizing chromosomal manipulations in sturgeons, particularly gynogenesis. The reasons of low survival rates were analyzed and the critical steps of gynogenesis induction processes were optimized. In addition, alternative ways of DNA inactivation in sperms were investigated, as well as the influence of native light-dependent DNA repair mechanisms on gynogenesis induction. Methods of interspecific gynogenesis usage for simplifying gynogenetic progeny separation were also proposed. Spectrophotometry analysis was used to investigate the ability of UV light, as the most common DNA inactivating agent, to penetrate into sperm. In addition, investigation of UV-irradiated sperm motility and results of partial gynogenesis induction showed that low transparency of sperms for UV-light can cause significant heterogeneity of UV-irradiation. As a result, a proper dilution of sperm was suggested as a critical step for homogeneous UV-irradiation of samples. Gynogenesis in sterlet was induced with chemical agents that damage sperm DNA, as an alternative to UV irradiation for applied in large-scale production of gynogenotes. All tested substances showed ability to inactivate DNA in spermatozoa, and thus producing gynogenotes. Negative impact of treatments with chemical agents on the sperm motility was observed. Subsequently, these treatments had a low efficiency of gynogenesis induction. The highest percentage of produced gynogenetic larvae 19.8 ? 8.9% was obtained by treatment with aminomethyl-4,5?,8-trimethylpsoralen (AMT) at 50 ?M followed by UV-A (360 nm) irradiation at dose of 900 J/m2. Therefore, this treatment could be used as a substitute for commonly used UV-C irradiation, e.g., in the case of large volumes of sperm. Detailed investigation of photoreactivation in sturgeon sperm revealed a significant level of light-dependent DNA restoration in sperms irradiated with high doses of UV-C light. Induction of gynogenesis with UV-C irradiation followed by exposure to visible light resulted in significant deviations from the typical Hertwig effect. In contrast, the red light with a wavelength of more than 600 nm did not result in decreased DNA damage, instead a moderate increase in damage was observed, i.e., it did not induce photoreactivation. Therefore, the use of infrared light to illuminate work stations during the induction of gynogenesis is suggested. The use of interspecific gynogenesis, particularly gametes of sturgeon species with different ploidy levels, was suggested as a way to simplify the separation of gynogenotes. In addition, application of this method allowed studying the effectiveness of DNA-inactivation and ploidy restoration treatments separately, as well as evaluation of fitness parameters and survival rates in each group of progeny without the physical separation of fish. Finally, the protocol for tetraploidization in sterlet was optimized for the prospective using tetraploid individuals for the induction of gynogenesis and androgenesis with diploid eggs and sperm. In conclusion, the described methods and protocols allowed gynogenesis induction in sturgeons with a survival rate sufficient for aquaculture, taking into consideration their high fertility, although further studies of the consequences of this treatment on fish is required.