In vitro fertilization (IVF) is fraught with problems and currently proteomics approaches are being tried out to examine the microenvironment of the follicle in order to assess biological and immunological parameters that may affect its development. Additionally, better understanding of reproductive process may help increase IVF birth rate per embryo transfer and at the same time avoid spontaneous miscarriages or life threatening conditions such as ovarian hyperstimulation syndrome. The primary aim of this study was to search for specific differences in protein composition of human follicular fluid (HFF) and plasma in order to identify proteins that accumulate or are absent in HFF. Depletion of abundant proteins combined with multidimensional protein fractionation allowed the study of middle- and lower-abundance proteins. Paired comparison study examining HFF with plasma/serum from women undergoing successful IVF revealed important differences in the protein composition which may improve our knowledge of the follicular microenvironment and its biological role. This study showed involvement of innate immune function of complement cascade in HFF. Complement inhibition and the presence of C-terminal fragment of perlecan suggested possible links to angiogenesis which is a vital process in folliculogenesis and placental development. Differences in proteins associated with blood coagulation were also found in the follicular milieu. Several specific proteins were observed, many of which have not yet been associated with follicle/oocyte maturation. These proteins together with their regulatory pathways may play a vital role in the reproductive process.

The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

Regulation of mRNA translation by cytoplasmic polyadenylation is known to be important for oocyte maturation and further development. This process is generally controlled by phosphorylation of cytoplasmic polyadenylation element binding protein 1 (CPEB1). The aim of this study is to determine the role of Aurora kinase A in CPEB1 phosphorylation and the consequent CPEB1-dependent polyadenylation of maternal mRNAs during mammalian oocyte meiosis. For this purpose, we specifically inhibited Aurora kinase A with MLN8237 during meiotic maturation of porcine oocytes. Using poly(A)-test PCR method, we monitored the effect of Aurora kinase A inhibition on poly(A)-tail extension of long and short cyclin B1 encoding mRNAs as markers of CPEB1-dependent cytoplasmic polyadenylation. Our results show that inhibition of Aurora kinase A activity impairs neither cyclin B1 mRNA polyadenylation nor its translation and that Aurora kinase A is unlikely to be involved in CPEB1 activating phosphorylation.

The multiprotein complexes known as condensins (I and II) are major players in chromosome dynamics in mitotic and meiotic cells. Here, we report for the first time the detection of different condensin subunits from both complexes in mammalian oocytes. Using immunoblotting analysis we examined expression levels of condensin subunits during meiotic maturation of porcine oocytes. The expression of the core subunit structural maintenance of chromosomes 2 (SMC2), identical in both condensin complexes, did not change significantly during maturation. Similarly, there was no significant change in the expression of the chromosome associated protein (CAP)-H and CAP-H2 subunits, components of condensin I and II, respectively. Conversely, the expression profiles of CAP-G, CAP-D2 (condensin I) and CAP-D3 (condensin II) were more interesting. At least two isoforms of the CAP-D2 subunit were detected, along with three isoforms of the CAP-D3 and CAP-G subunits. We suggest that this diverse migration of subunit isoforms is due to post-translational modification. Earlier, it was reported that non-SMC proteins are phosphorylated by cyclin-dependent kinase 1. In the present study, we analysed the phosphorylation status of the three subunits in oocyte extracts using alkaline phosphatase treatment and we found that at least the fastest migrating form of CAP-D3 was likely to be phosphorylated in maturing porcine oocytes. In addition, the localisation of CAP-H and CAP-H2 subunits was examined using immunofluorescence staining with specific antibodies, as well as following microinjection of their enhanced green fluorescent protein-tagged mRNA into germinal vesicle-stage oocytes. CAP-H was found in the cytoplasm, whereas CAP-H2 was localised within the nucleus.