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The ability of oocytes and early cleavage-stage embryos to regulate their volume is essential to avoid developmental arrests at in vivo-osmolarities. This is accomplished primarily via GLYT1-mediated glycine transport into the cells. GLYT1 activity has previously been shown to be absent in freshly isolated oocytes but becomes activated ~3-4 hours after oocyte maturation has been initiated either by isolation from ovarian follicles in vitro or following an ovulatory stimulus in vivo. GLYT1 activity then persists until the 4-cell stage of preimplantation embryo development. GLYT1 has been shown to spontaneously activate in oocytes that are isolated from follicles either as denuded oocytes or as cumulus-oocyte complexes (COCs), this implies that GLYT1 activity is suppressed in intact follicles in the ovary. However, it is not known how GLYT1 activity is suppressed within the ovarian follicle or how initial GLYT1 activation occurs. The activation of independent cell volume regulation in oocytes first involves the release of the strong adhesion between the oocyte and zona pellucida (ZP) followed by secondary GLYT1 activation. These two processes have been shown to occur spontaneously in fully grown oocytes following isolation from ovarian follicles, however, it is not known whether small growing oocytes within ovarian follicles already possess the ability to detach from the ZP and activate GLYT1. An osmotic assay was used to determine when during oogenesis oocytes are first able to detach from the ZP while the ability to activate GLYT1 was determined by measuring [3H]-glycine uptake into oocytes. I found that oocytes acquire the ability to detach from the ZP when they are nearly fully grown and similarly, that high levels of GLYT1 activity first develop in isolated oocytes during the late stages of oogenesis. Furthermore, I showed that SLC6A9 protein (GLYT1 transporter protein) and Slc6a9a transcripts steadily increased during oogenesis with SLC6A9 protein becoming localized to the oocyte plasma membrane during oocyte growth with predominant membrane localization apparent in fully grown oocytes. Taken together, these results suggest that oocytes become able to detach from the ZP and fully activate GLYT1 towards the end of oogenesis but that these processes remain suppressed in the ovarian follicle. Intact and punctured antral follicles were used as a model to examine the potential mechanism(s) mediating GLYT1 suppression before ovulation is triggered. Using these models, I found that GLYT1 activity remains suppressed within preovulatory antral follicles in contrast to the spontaneous GLYT1 activation that occurred in isolated denuded oocytes or within COCs. Recently, the mechanism mediating oocyte maintenance of prophase I arrest within the ovarian follicle was elucidated and was shown to depend on the release of Natriuretic Peptide Precursor C (NPPC) from mural granulosa cells (MGCs) into follicular fluid which binds to NPR2 guanylate cyclases on cumulus cells stimulating the production of cyclic GMP (cGMP) within these cells. Diffusion of cGMP from cumulus granulosa cells to the oocyte via gap junctions is required to maintain meiotic arrest. Although GLYT1 activation and meiotic resumption are both suppressed in antral follicles prior to the ovulatory trigger and these two processes occur simultaneously following oocyte isolation from ovaries, I have shown here that GLYT1 suppression within the preovulatory antral follicle is mediated by a mechanism distinct from the gap junction-dependent NPPC-cGMP pathway controlling meiotic arrest. I also showed for the first time a direct requirement for meiotic arrest of both gap junctions between granulosa cells (composed of connexin-43) and between the inner layer of cumulus granulosa cells and the oocyte (composed of connexin-37). Since I showed that GLYT1 was suppressed in isolated antral follicles but not COCs, I hypothesized that MGCs are required to maintain low GLYT1 activity in antral follicles. I showed here that MGCs isolated from preovulatory antral follicles were sufficient to maintain GLYT1 suppression in co-cultured COCs, but not denuded oocytes. Furthermore, I found that GLYT1 activity was suppressed in COCs cultured in conditioned medium from MGC cultures. Thus, GLYT1 activity appears to be suppressed within the ovary prior to the ovulatory LH-stimulus likely by an unidentified inhibitory signal within the ovarian follicle originating from the MGCs and propagated by a gap junction-independent mechanism involving multiple cell types in the follicle.

Meiotic division of a female germ cell, an oocyte, is more prone to segregation errors and consequently to aneuploidies than meiosis of a sperm. Aneuploidies and chromosomal aberrations in oocytes increase with higher maternal age in humans and also in mice. Meiotic maturation onset is connected with activity of cyclin dependent kinase 1 (CDK1) that leads to dissociation of nuclear membrane. Moreover regulation of translation of key transcripts is necessary for proper meiotic progression. In thesis findings from four scientific publications are interpreted. We have analyzed the timing of nuclear envelope breakdown (NEBD) and polar body extrusion in mouse oocytes originating from two distinct female age groups: young (2 months old) and aged (12 months old). We found that meiotic maturation happens faster in aged females' oocytes due to early phosphorylation of Lamin A/C, a component of nuclear lamina, and rapid dissociation of nuclear membrane. Moreover aged females' oocytes presented unique characteristic invaginations of nuclear membrane and thus significantly increased circumference of the nuclear envelope compared to the oocytes from young females. These data combined with increased activity of CDK1 and Cyclin B, as well as increased translation of factors that regulate the translation itself,...

Bisphenol A is a widely used chemical in the manufacture of plastics. The presence of BPA in the environment adversely affects human health due to contamination of air, drinking water and food. Growing concerns about the effects of BPA have led to its regulation in production and development of alternative chemicals to BPA, such as bisphenol S (BPS). However, the effects of BPS were not properly tested before its introduction to production and the effects on human reproduction are still unknown. For this reason, it is desirable to test the effect of BPS on mammalian oocyte development. This study hypothesizes that BPS exposure causes inhibition of meiotic maturation of porcine oocytes in vitro. This study aims to investigate the potency of BPS at low concentrations corresponding to normal human exposures to selected porcine oocyte proteins. The results of this study demonstrate the negative effect of BPS on the progression of meiotic maturation and reaching the mature oocyte stage. In addition, the results show an increase in the formation of defective meiotic spindles and a disruption of mitochondrial integrity after exposure to BPS concentrations. However, the effect of BPS on double-strand breaks was not demonstrated in this study, in contrast to the case of BPA. Taken together, the results show...

The global DNA methylation pattern is erased and re-established during oogenesis and again in preimplantation (PI) embryo development. Understanding where these methyl groups come from and how the process of methylation is regulated is important, as disruptions could result in detrimental effects. The methionine cycle that produces the cellular methyl pool is linked to the folate cycle. The key enzyme linking theses cycles is Methylenetetrahydrofolate Reductase (MTHFR) which converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. Mthfr RNA and protein are present throughout mouse oocyte and PI embryo development, including the germinal vesicle, MII egg, 1-cell embryo, 2-cell embryo, morula and blastocysts. In MII eggs the protein appears to be heavier than in any other stage. This was reversed by treatment with Lambda Protein Phosphatase (LPP), indicating that MTHFR is phosphorylated in MII eggs. MTHFR was progressively phosphorylated beginning shortly after initiation of meiotic maturation, reaching maximal levels in MII eggs before decreasing after egg activation using strontium chloride. Potential kinases responsible for the phosphorylation of MTHFR have been identified however not in oocytes or PI embryos. DYRK1A/1 and GSK3A/B have both been suggested to mediate the phosphorylation, however when inhibited showed no effect on the oocyte sample. An LC-MS/MS assay was attempted to measure the activity of MTHFR in wildtype and knockout mouse liver samples, however unsuccessful in the amounts needed to be used for comparison to oocytes. Overall, MTHFR is present in the developing stages of interest and is mediated in some capacity by phosphorylation modifications around the MII stage of development.

During early embryonic development oocytes are arrested in prophase I of the first meiotic division, in which they can persist for years. After reaching sexual maturity and the luteinizing hormon surge resumption of meiosis and meiotic maturation occur. Oocytes are arrested again at metaphase of the second meiotic division. At this stage they are ovulated and waiting for a fertilisation. Oocytes are during their development exposed to factors that cause DNA damage, of which DNA double-strand breaks (DSBs) are the most serious threat. The maintaining of genome integrity is crucial for quality of oocytes, fertility and proper embryonic development. The mechanism of the oocyte response to DSBs presence is not fully understood and it seems to differ from somatic cells. We assume that DSBs are repaired during meiotic maturation probably by a mechanism of homologous recombination (HR). In this thesis we focuse on essencial recombinase RAD51, which participates in the repair by HR. We found that RAD51 inhibition leads to an increase of segregation errors in anaphase I. Using high resolution live cell imaging we observed chromosomal fragments and anaphase bridges. Immunofluorescence detection of DSBs-marker γH2AX showed increased amount of DSBs in prophase I and MII stage after RAD51 inhibition. Our data...