1,721,100 research outputs found
Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation
No full textCharacterization of two types of prostasomes with distinct molecular compositions
No full textDuring the last years it has become evident that many different cell types can communicate with each other through intercellular transfer of extracellular vesicles. Such cell-derived membrane vesicles function in several physiological processes and also in disease. This thesis describes two subclasses of prostasomes, extracellular vesicles that are released by prostate epithelial cells and ultimately enter the seminal plasma. Prostasomes stimulate sperm cell motility and influence sperm cell capacitation and the acrosome reaction, all of which are prerequisites for successful fertilization. Furthermore, prostasomes suppress immune-mediated destruction of sperm cells in the female reproductive tract. However, the exact molecular mechanisms through which prostasomes exert such diverse functions remain largely unknown. In the studies described in this thesis, prostasomes were purified and their protein, lipid and nucleic acid contents characterized. This resulted in the definition of two distinct types of prostasomes, a smaller (50 nm) and a larger (100 nm) type, which may perform different functions. The two prostasome types have distinct protein and lipid compositions. Many other types of extracellular vesicles are known to contain RNA, which may elicit epigenetic effects in their target cells. However, the RNA content of prostasomes had not been explored. In the research described in this thesis, RNA was found associated with the large prostasomes. Deep sequencing of this RNA showed that the majority is composed of a type that had not been described before. Physiological target cells for the larger prostasomes, and hence for their RNA contents, remained unknown. Given its unique composition, prostasomal RNA may serve as a biomarker of prostate cancer. The blood of healthy men is devoid of prostasomes, but in case of prostate cancer prostasomes may reach the blood stream as well. Comparison of prostasomal RNA from prostate cancer patients with that of healthy men, either from blood, urine or seminal plasma, may provide new insights in its potential as cancer biomarker. The interactions between small prostasomes and sperm cells during sperm cell capacitation were studied in an equine model. Small prostasomes were found to bind to sperm cells, but only at conditions that favoured sperm cell capacitation. Moreover, recruited prostasomes reduced the degree of protein tyrosine phosphorylation in sperm cells, which is a hallmark of a late phase in sperm cell capacitation. Prostasomes may therefore help to prevent premature full capacitation of sperm cells and their acrosome reaction. Other studies showed that transfer of progesterone receptors to the sperm cells through fusion of prostasomes with their plasma membrane. Once the sperm cells have reached the progesterone-secreting cumulus cells that surround the oocyte, transfer of progesterone receptors by prostasomes may assist the progesterone-dependent stimulation of sperm cell hypermotility and the acrosome reaction. Thus, prostasomes may bind and accompany sperm cells on their journey to the oocyte, and fuse with the sperm cells only upon their approach of an oocyte, providing them with tools that are required for effective oocyte fertilizatio
Identification and Characterization of Adult Porcine Muscle Stem Cells
No full textIn the past decade, tissue-specific stem cell research has been emerging. Stem cells are characterized by a long-term expansion and a broad developmental potential in vitro. Pre-clinical studies appear promising, but still many limitations have to be overcome before broad therapeutic use of stem cells is save and efficient. Meanwhile, the use of stem cells for drug screening and toxicology studies are also beneficial to biomedical science. To cure muscular dystrophy no efficient therapy exists. Stem cells can be used to replace the affected tissue with newly formed fibers. Besides biomedical applications, stem cell technology can also be used for the generation of cultured meat for human consumption (‘in vitro meat’). By this we can offer an alternative to the bio-industry by using stem cell technology for large expansion of farm animal-derived stem cells and subsequently the formation of muscle tissue in an in vitro culture system. In this PhD project we observed that muscle tissue is a rich source of various stem and progenitor cell types. We optimized stem cell isolation protocols and developed methods for the characterization for porcine muscle stem cells. Thereby, we increased our knowledge concerning the regulation of stem cell maintenance and their development potential. By optimization of the in vitro culture conditions for porcine muscle stem cells we provide a cell culture system in which we improved the maintenance of the stem cell characteristics. Furthermore, based on protein expression we showed that we could select for a highly myogenic stem cell population from porcine muscle tissue. Overall, this knowledge will facilitate the development of stem cell therapy for regenerative medicine, the use of stem cells in toxicology screening and for the generation of in vitro cultured meat
Intra-follicular interactions affecting mammalian oocyte maturation
No full textNuclear oocyte maturation is defined as reinitiation and progression of the first meiotic division and subsequently formation of the methaphase II (MII) plate. Concomitantly with nuclear maturation, cytoplasmic maturation which is essential for proper fertilization and early embryo development is completed. The trigger for resumption of meiosis in vivo is considered to be the preovulatory surge of luteinizing hormone (LH), whereas oocytes removed from their follicle spontaneously resume meiosis in vitro and mature up to the MII stage. For oocyte collection in women, ovarian stimulation protocols involve gonadotrophin-releasing hormone agonists or antagonists in combination with gonadotrophins to generate multiple follicles in the ovary containing in vivo matured oocytes. Recovery of immature oocytes followed by in vitro maturation of these oocytes is an attractive alternative for this comprehensive stimulation protocol. Although maturation in vitro can be accomplished in human oocytes, like in cattle it is associated with reduced developmental competence. Fundamental studies on the process of maturation in vitro as well as in vivo may therefore ameliorate culture conditions for the maturation of oocytes in vitro. The data presented in chapter 2, 3 and 4 of this thesis confirm the important role of granulosa and theca cells in preventing spontaneous or gonadotrophin triggered resumption of meiosis in bovine oocytes originating from small and medium sized follicles. This knowledge can contribute to the design of two-step culture systems leading to improved conditions during final oocyte maturation. Already since the 1950s there are reports about disrupted reproduction functions after severe changes in energy balance resulting in suboptimal body conditions. At present it has been recognized that dairy cows experience reduced fertility when nutrient requirements for maintenance and lactation exceed nutrient intake. Moreover, in women undergoing IVF treatment both overweight and underweight lead to decreased chances of pregnancy. Selection for the trait of milk production and the associated reduced fertility in the modern dairy cow and the alarming increase in the proportion of obese women are two examples that demonstrate the demand for research on pathways linking energy balance and reproduction. Data presented in chapter 5 and 6 of this thesis concerning the role of leptin, one of the factors connecting energy balance and reproduction, in oocyte maturation and subsequent embryo production point to an enhancing effect of leptin on oocyte maturation. However high leptin levels in follicular fluid from patients at the day of oocyte retrieval are related to a lower chance on pregnancy. Considering this discrepancy, further studies on the role of leptin in reproduction should focus on early embryonic development and implantation. In general the studies presented in this thesis provide more insights on the fundamental mechanisms regulating mammalian oocyte maturation with potential for development of improved methods of embryo production in vitro or for assisted reproduction applications in livestock and humans
Veterinary applications of induced pluripotent stem cells: Regenerative medicine and models for disease?
No full textInduced pluripotent stem cells (iPSCs) can now be derived from a tissue biopsy and represent a promising new platform for disease modelling, drug and toxicity testing, biomarker development and cell-based therapies for regenerative medicine. In regenerative medicine, large animals may represent the best models for man, and thereby provide invaluable systems in which to test the safety and the potential of iPSCs. Hence, testing iPSCs in veterinary species may serve a double function, namely, developing therapeutic products for regenerative medicine in veterinary patients while providing valuable background information for human clinical trials. The production of iPSCs from livestock or wild species is attractive because it could improve efficiency and reduce costs in various fields, such as transgenic animal generation and drug development, preservation of biological diversity, and because it also offers an alternative to xenotransplantation for in vivo generation of organs. Although the technology of cellular reprogramming using the so-called ‘Yamanaka factors’ is in its peak expectation phase and many concerns still need to be addressed, the rapid technical progress suggests that iPSCs could contribute significantly to novel therapies in veterinary and biomedical practice in the near future. This review provides an overview of the potential applications of iPSCs in veterinary medicine
Archiving biomedical mouse models by ovary cryopreservation
No full textThe use of genetically modified (GM) mice for biomedical research has tremendously increased since 1980’s. Housing the rapidly expanding number of mouse lines has become a problem and specific lines were even accidentally lost due to the lack of space or loss of fertility during breeding for maintenance. Cryopreservation is a strongly recommended approach to solve these problems, also from an ethical point of view. There are also technical risks by small-scale breeding, including genetic drift, microbiological or genetic contamination, disease, or large-scale disasters. The investigations presented in this thesis focus on cryopreservation of mouse ovaries to maintain the expanding populations of GM mice, and the target strains (C57BL/6, FVB, BALB/c) utilized are those commonly used as the genetic background. The major aim of the research is to develop an efficient, economic and ethically sound cryopreservation and validation protocol for archiving the mutant mouse lines. In chapter 2, a comparison is made of the currently used technologies for cryopreservation of germplasm in mice and the subsequent retrieval of these mutant lines. Detailed managerial information concerning animal use, and time and cost related to these technologies is evaluated. In chapter 3, a cryopreservation protocol for mouse ovaries using high concentrations of cryoprotectants and instrumented ultra-rapid freezing for vitrification is developed and validated by ovary transplantation and subsequent breeding. The robustness of the developed freezing protocol is investigated from various genetically modified lines with different genetic backgrounds (chapter 4). The over-time functionality of the transplanted ovaries is investigated with regard to efficiency in delivering mutant offspring. In chapter 5, the in vivo development of cryopreserved ovarian tissue is investigated by in vivo imaging after allotransplantation. The optimal donor age is also evaluated. The use of a spontaneous albino mutant of the (black) C57BL/6 as a recipient is also evaluated as a rapid method for identifying the parentage of delivered pup. In chapter 6, the application of transplantation of fresh grafts is evaluated in propagating a subfertile mutant mouse line and the reduced reproduction efficiency related to a particular genetic modification is investigated. Specific attention is focused on the origin of the abnormality. Finally, an overview of the results presented in these studies and their possible implications for future practice and researches are discussed in chapter 7. “There is really no limit to the number of mutant mouse strains that could be generated and expanded to infinity” is practically true in the filed of mouse genetics. Thus, how to manage the mouse colonies in a responsible way is an urgent issue and is an obligated responsibility of researchers. In this thesis, we present the practical information about cryopreservation of germplasm for the management of mutant mouse colonies. The successful application on cryopreservation of juvenile ovaries provides an instant solution to animal facilities that are facing space problems of housing mouse colonies and may also provide useful information to cryopreservation of human ovarian tissue, serving as a model for autotransplantation and studying graft/host interactions in a variety of models for human diseases
SNARE protein mediated secretory events in porcine fertilization
No full textFertilization is an event that involves highly dynamic intra- and intercellular processes which enable the merge of two gametes with the aim to ultimately form a new organism. Preparative changes are required in both gametes prior to fertilization since freshly ejaculated sperm cells and germinal vesicle (GV) stage arrested oocytes are not capable to form a zygote. Only functionally matured sperm can fertilize the oocyte and specific modifications have to be accomplished at the surface of the sperm head. Like sperm, the oocyte has to be prepared for gamete fusion and to this end resumes meiosis prior to ovulation. Immediately after sperm binding to the oocyte plasma membrane (oolemma), the oocyte initiates a restrictive process - the cortical reaction (CR) - to prevent polyspermic fertilization which is incompatible with subsequent normal development of the zygote. Aberration of any of these processes will lead to the failure of fertilization; therefore, the aim of this thesis is to contribute to the knowledge of specific intracellular processes (namely the acrosome reaction in the sperm cell and the CR in the oocyte) that are crucial in gametes for successful fertilizatio
Fibroblast growth factor-2 expression in the preimplantation equine conceptus and endometrium of pregnant and cyclic mares
No full textUterine-derived growth factors and cytokines play essential roles in regulating preimplantation conceptus development. In several species, fibroblast growth factor-2 (FGF2) promotes embryogenesis, trophoblast cell migration, and adhesion. This study investigated mRNA expression for FGF2, its receptors (FGFR1-4), the activating factor FGF binding protein (FGF-BP) in equine endometrium and trophectoderm during early pregnancy and the estrous cycle, and localized FGF2 protein in both endometrium and conceptus tissues. FGF2, FGFRs1-4, and FGFBP mRNAs were expressed in endometrium throughout the estrous cycle and early pregnancy, and in days 14 to 28 conceptus membranes. FGF2 transcription was higher during estrus than on days 7 or 14 of diestrus, suggesting estrogen dependency. Endometrial expression of FGF2 mRNA and protein increased as pregnancy progressed from days 21 and day 28; FGF2 protein was localized predominantly in the luminal and glandular epithelium. FGF2 mRNA was detectable in trophectoderm from as early as day 14, and transcription and translation increased in day 21 and 28 allantochorion. FGF2 protein was localized mainly in the trophectoderm up to day 21 but was present in both trophectoderm and endoderm of day 28 allantochorion. FGFR1 mRNA was down-regulated in the endometrium at day 7 of diestrus but increased again by day 14. Gene expression for all of the FGFR2 splice variants, including FGFR2IIIc, was up-regulated during estrus. During early pregnancy, endometrial FGFR1 expression decreased, whereas FGFR2IIIc expression did not change. Conceptus mRNA expression for all FGFRs increased as pregnancy progressed. FGFBP expression remained unchanged in endometrium, but increased in the conceptus between days 14 and 28, suggesting a role in regulating FGF2 activity in the developing conceptus. We conclude that during weeks 3 and 4 of pregnancy, the equine endometrial epithelium produces FGF2, which may play a role in trophoblast development and adhesion
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