The effects of adding the extracellular glycosaminoglycans (GAGs), hyaluronic acid (HA) and chondroitin sulphate (CS) to rat in vitro fertilisation (IVF) media were assessed. Metaphase II (MII) oocytes were also incubated in GAG-supplemented modified rat 1-cell embryo culture medium (mR1ECMCBSA) for 3 days. Cytoplasmic fragmentation was significantly reduced in mR1ECMCBSA with HA (39.0-48.0%) compared with the control (82.0%). In IVF experiments, neither HA (8.0-30.8%) nor CS (9.7-42.5%) improved fertilisation rates compared with controls fertilised in M16 (47.2%) or enriched Krebs-Ringer bicarbonate solution (61.5%). RT-PCR and Western blot were used to probe for CD44 mRNA and protein in Sprague-Dawley gametes and cumulus cells. CD44 was identified in cumulus cells, suggesting a role for oocyte maturation and cumulus expansion. The CD44 protein was also present on caudal epididymal spermatozoa that were highly stimulated by CS in vitro implicating a role in fertilisation for CS and CD44.

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.

In vitro manipulation of the murine embryo has advanced over the last 50 years with the introduction of ICSI, knockout and cloning technologies. Yet the same technologies have not developed in the rat despite high similarities between proteins with a function in fertilization and fundamental differences seem apparent. A robust in vitro culture medium, mR1ECM capable of supporting pre-implantation development through to the blastocyst stage has only recently emerged. In vitro culture (IVC) of in vivo fertilized embryos in mR1ECM with PVA showed 28.6 % of zygotes and 79.3 % of 2-cell embryos could develop to blastocyst. The major problem was in the initial division after collection. Almost half the failure to develop occurred at this point. KSOM proved to be significantly worse than mR1ECM+PVA for IVC with arrest prominent at the 2-cell stage (89.8%). mR1ECM even supported limited hatching of blastocysts from the zona pellucida (ZP). In vitro fertilization (IVF) with mR1ECM has not been successful. This may be due to failure of sperm to undergo capacitation. We unsuccessfully attempted to induce capacitation over a range (0.5-6.0 h) of preincubation times. A major difficulty is that a characterised marker for capacitation in the rat has not been identified. Another problem for successful IVF is that the majority of oocytes collected underwent spontaneous activation resulting in arrest and an oocyte incapable of fertilization (78.6%). This was characterised by cortical granule exocytosis and an alteration to the ZP molecular structure, which precludes sperm binding to the ZP. These significant limitations need to be overcome if we are to be able to study interaction between rat sperm and egg at the molecular level. The overall goal of this project is to be able to identify and characterise oolemma proteins that interact with sperm counterparts as the two gametes undergo fusion.

Proprotein convertase 6 (PC6), is a key player during embryo implantation in humans and mice. We have previously shown that PC6 is essential for decidualisation in the mouse and knockdown of endometrial PC6 leads to implantation failure. The PC family of proteases, including PC6, are necessary for transmission of human immunodeficiency virus (HIV). It has been postulated that inhibition of PC activity could prevent HIV infection. We hypothesise that PC6 is a prospective target for the development of a dual role contraceptive for women to avoid pregnancy and protect from HIV infection. The aim of this study is to evaluate if a PC6 inhibitor that is capable of preventing HIV transmission can block implantation in mice. We used a generic PC peptide substrate to assess the potency of the inhibitor to block PC6 activity in vitro. The substrate releases a fluorochrome when cleaved by PC6; no fluorescent signals were observed in samples when inhibitor concentrations were 10μM or higher. We then gauged inhibitor uptake by the uterus over 24 h in mice by two delivery routes; intrauterine injection (IU) and vaginal delivery (VD) with a neutral gel. Uptake was tracked with a FITC-conjugated inhibitor at 50μM (IU) and 500μM (VD). Strong fluorescent signals were seen at 2, 4, 6 and 24 h at sites of endometrial PC6 activity in the IU and VD groups. Administration of a 50μM dosage (20μl) to the uterine lumen (IU) caused a significant reduction (P = 0.002) in the number of implantation sites compared with controls (saline only) when treated between 2000–2100 on E3.75. The inhibitor's ability to block uterine PC6 activity and implantation via VD was assessed and to date outcomes have suggested that correct timing is crucial to prevent implantation and decidualisation. These outcomes show the potential of the inhibitor to block implantation in mice.