Many primate populations face the threat of extinction due to habitat loss, intensive agriculture, hunting for meat, the pet trade and/or use in traditional medicines. An alternative approach to in situ conservation includes gene banking and the use of assisted reproductive technologies (ART), such as oocyte in vitro maturation (IVM) and in vitro fertilization (IVF). Although many of these 'high-tech' solutions have not yet been proven viable for pragmatic wildlife conservation, basic research and development of these emerging tools can provide necessary information needed to optimize these techniques and institute ART as a routine practice in conservation efforts. A severely limiting factor in the successful application of ARTs is the availability of mature developmentally competent oocytes. Oocyte maturation involves many nuclear and cytoplasmic factors, which can be affected by maturation conditions and female age. In vitro maturation does not have the same success rate across species studied. In primates especially, IVM oocytes exhibit reduced developmental capacity upon fertilization when compared to in vivo matured (IVO) oocytes. This study aimed to investigate possible causes of reduced developmental capacity of primate IVM oocytes using the rhesus macaque (Macaca mulatta) as a model. Research efforts included investigation of ovarian senescence, oocyte karyotype and spindle morphology, and establishment of an optimal sperm cryopreservation protocol for use in IVF. Histological examination of the rhesus ovary demonstrated an age-related pattern of follicle depletion similar to that described in the human ovary. Oocyte karyotype analysis revealed a significant effect of IVM on the frequency of hyperhaploidy. In addition, immunostaining and confocal microscopy demonstrated a significant increase of anomalous chromosome congression on the oocyte metaphase II spindle equator in relation to IVM and donor female age. These results indicate that IVM can produce serious, if not lethal consequences for embryo development. This study presents baseline data on ovarian aging in the rhesus macaque and aspects of nuclear maturation during macaque IVM that may contribute to the design of primate oocyte recovery plans. Implementation of either of two sperm cryopreservation methods originally developed for rhesus and vervet monkeys will aid future investigation of the developmental capacity of IVM oocytes.

Die Metaphasenspindel ist eine selbstorganisierende molekulare Maschine, die die entscheidende Funktion erfüllt, das Genom während der Zellteilung gleichmäßig zu trennen. Spindellänge und -form sind emergente Eigenschaften, die durch komplexe Wechselwirkungsnetzwerke zwischen Molekülen hervorgerufen werden. Obwohl erhebliche Fortschritte beim Verständnis der einzelnen molekularen Akteure erzielt wurden, die ihre Länge und Form beeinflussen, haben wir erst kürzlich damit begonnen, die Zusammenhänge zwischen Spindelmorphologie, Dynamik und Materialeigenschaften zu untersuchen. In dieser Arbeit untersuchte ich zunächst quantitativ die Rolle zweier molekularer Kraftgeneratoren - Kinesin-5 und Dynein - bei der Regulierung der Spindelform von Xenopus-Eiextrakt. Eine Störung ihrer Aktivität veränderte die Spindelmorphologie, ohne die Gesamtmasse der Mikrotubuli zu beeinflussen. Um die Spindelform physikalisch zu stören, wurde ein Optical Stretcher (OS) -Aufbau entwickelt. Obwohl das OS Vesikel in Extrakten verformen könnte, konnte keine Kraft auf Spindeln ausgeübt werden. Die Untersuchung des Brechungsindex der Struktur mittels optischer Beugungstomographie (ODT) ergab, dass es keinen Unterschied zwischen Spindel und Zytoplasma gab. Korrelative Fluoreszenz- und ODT-Bildgebung zeigten, wie sich die Materialeigenschaften innerhalb verschiedener Biomoleküle räumlich unterschieden. Die Gesamttrockenmasse der Spindel skalierte mit der Länge, während die Gesamtdichte konstant blieb. Interessanterweise waren die Spindeln in HeLa-Zellen dichter als das Zytoplasma. Schließlich deckte eine störende Mikrotubulusdichte auf, wie die Gesamttubulinkonzentration die Spindelgröße, die Gesamtmasse und die Materialeigenschaften regulierte. Insgesamt bietet diese Studie eine grundlegende Charakterisierung der physikalischen Eigenschaften der Spindel und hilft dabei, Zusammenhänge zwischen der Biochemie und der Biophysik einer aktiven Form weicher Materie zu beleuchten. / The metaphase spindle is a self-organising molecular machine that performs the critical function of segregating the genome equally during cell division. Spindle length and shape are emergent properties brought about by complex networks of interactions between molecules. Although significant progress has been made in understanding the individual molecular players influencing its length and shape, we have only recently started exploring the links between spindle morphology, dynamics, and material properties. A thorough analysis of spindle material properties is essential if we are to comprehend how such a dynamic structure responds to forces, and maintains its steady-state length and shape. In this work, I first quantitatively investigated the role of two molecular force generators– Kinesin-5 and Dynein in regulating Xenopus egg extract spindle shape. Perturbing their activity altered spindle morphology without impacting total microtubule mass. To physically perturb spindle shape, an Optical Stretcher (OS) setup was developed. Although the OS could deform vesicles in extracts, force could not be exerted on spindles. Investigating the structure’s refractive index using Optical Diffraction Tomography (ODT) revealed that there was no difference between the spindle and cytoplasm. Correlative fluorescence and ODT imaging revealed how material properties varied spatially within different biomolecules. Additionally, spindle mass density and the microtubule density were correlated. The total dry mass of the spindle scaled with length while overall density remained constant. Interestingly, spindles in HeLa cells were denser than the cytoplasm. Finally, perturbing microtubule density uncovered how total tubulin concentration regulated spindle size, overall mass and material properties. Overall, this study provides a fundamental characterisation of the spindle’s physical properties and helps illuminate links between the biochemistry and biophysics of an active form of soft matter.