Molecular and morphological studies of folliculogenesis, oocyte maturation and embryogenesis in humans

Abstract: Infertility is a problem that affects about 15 percent of all couples. Many can be helped by assisted reproductive techniques, but still today many couples are treated without a successful outcome. The reason for infertility is sometimes known, but there are also many cases of unexplained infertility. Therefore, basic knowledge about follicle development, maturation of oocytes and preimplantation embryo development needs to be explored further. In article I we investigated the possibility to quantify gene expression in single embryos and blastomeres to detect the expected increase occurring at the time for the embryonic gene activation (EGA). We found that real-time RT-PCR was sensitive enough for this purpose, as we could detect an increased gene expression of the translation initiation factor 1A (eIF-1A) at the expected time in mouse and human preimlantation embryos and in mouse blastomeres. Possibly, with further improvements such measurements could be useful when evaluating the developmental capacity of early embryos. In article II we utilized the unique opportunity to study non-luteinized granulosa cells (GC s) from in vitro maturation (IVM) treatments. We studied the effect of gonadotrophins (FSH and hCG) on the non-luteinized GCs by measuring secretion of estrogen and progesterone and the expression levels of the enzymes p450aromatase and p450scc (side chain cleavage enzyme) before and after treatment. These measurements were compared to luteinized GCs from conventional IVF-treated patients. Furthermore, we studied how the receptors for LH and FSH reacted to the different treatments in nonluteinized GC s. We observed increased or unchanged hormone production and gene expression of the enzymes involved in their synthesis in the non-luteinized GC s, while in the luteinized GC s the hormone production and enzyme expression was decreased or unchanged in response to the different treatments. In IVM GC s the LH receptor expression increased with FSH treatment but the expression of the FSH receptor was unaffected. We concluded that the more reactive non-luteinized GC s are more interesting to study as they are still in a stage where they can influence the oocyte. The effect of growth differentiation factor -9 (GDF-9) on follicular development was investigated in article III. This was studied in ovarian tissue cultures using different GDF-9 agonists and antagonists. We found that the addition of exogenous rhGDF-9 stimulates growth of early follicles and promotes the transition from the primary to the secondary stage. We also saw that this transition could be inhibited by blocking endogenous GDF-9 using the soluble receptor BMPRII-Fc. These findings can be very useful trying to mature follicles from cryopreserved tissue in vitro. Finally, in article IV we have adapted a recently developed research instrument, the Cell- IQ®, for studies of oocyte maturation in vitro. In this instrument the incubator and the imaging equipment is integrated and we can follow the different maturation events in detail without disturbing the culture conditions. This offers an excellent opportunity to optimize the culture conditions in order to improve the clinical IVM procedure. Our findings provide new information regarding the complex regulation of folliculogenesis and oocyte maturation and we also provide helpful tools for further investigations aiming at improving assisted reproductive techniques.