Identification of a CIP4 PKA phosphorylation site involved in the regulation of cancer cell invasiveness and metastasis.

CDC42 interacting protein 4 (CIP4) is a CDC42 effector that coordinates membrane deformation and actin polymerization. The correlation of CIP4 overexpression with metastatic capacity has been characterized in several types of cancer. However, little information exists on how CIP4 function is regulated. CIP4 interacts with A-kinase (PKA) anchoring protein 350 (AKAP350) and CIP4 is also a PKA substrate. Here, we identified CIP4 T225 as the major CIP4 PKA phosphorylation site. In vitro and in vivo experiments using hepatocellular carcinoma (HCC) and breast cancer cells showed that expression of a CIP4(T225E) phosphomimetic mutant increased cancer cell metastatic capacity and that, conversely, expression of a CIP4(T225A) non-phosphorylatable mutant reduced invasive properties. PKA inhibition decreased to CIP4(T225A) cell-levels control but not CIP4(T225E) cell migratory and invasive efficiency. Concomitantly, our studies indicate that CIP4 T225 phosphorylation promotes the formation of functional invadopodia and enhances CIP4 localization at these structures. Our findings further provide mechanistic data indicating that CIP4 T225 phosphorylation facilitates CIP4 interaction with CDC42. Altogether this study identifies a signaling pathway that involves CIP4 phosphorylation by PKA during the acquisition of a metastatic phenotype in cancer cells.

Tissue plasminogen activator (tPA) of paternal origin is necessary for the success of in vitro but not of in vivo fertilisation in the mouse.

Besides its fibrinolytic function, the plasminogen-plasmin (PLG-PLA) system is also involved in fertilisation, where plasminogen activators bind to plasminogen to produce plasmin, which modulates sperm binding to the zona pellucida. However, controversy exists, depending on the species, concerning the role of the different components of the system. This study focused its attention on the role of the PLG-PLA system on fertilisation in the mouse with special attention to tissue plasminogen activator (tPA). The presence of exogenous plasminogen reduced invitro fertilisation (IVF) rates and this decline was attenuated by the presence of plasmin inhibitors in combination with plasminogen. The incubation of spermatozoa with either oocytes or cumulus cells together with plasminogen did not change the acrosome reaction but reduced the number of spermatozoa attached. When spermatozoa from tPA-/- mice were used, the IVF rate decreased drastically, although the addition of exogenous tPA during gamete co-incubation under invitro conditions increased fertilisation success. Moreover, fertility could not be restored after invivo insemination of tPA-/- spermatozoa in the female ampulla, although tPA-/- males were able to fertilise invivo. This study suggests a regulatory role of the PLG-PLA system during fertilisation in the mouse with possible implications in human reproduction clinics, such as failures in tPA production, which could be partially resolved by the addition of exogenous tPA during IVF treatment.

Optimization of post-cervical artificial insemination in gilts: Effect of cervical relaxation procedures and catheter type.

Post-cervical (pC) artificial insemination (AI) has been successfully developed for application in multiparous sows, although it has proved problematic in gilts. This study analyzes the use of pC-AI in gilts by two experiments. In the first experiment, the efficiency of pC-AI in gilts was evaluated using a multi-ring multiparous catheter (MpC), which led to 23.1% of the gilts being successfully inseminated. In gilts where insemination was not possible using an MpC, two alternatives were applied before a second attempt at insemination: 1) Vetrabutin Chlorhydrate (VC) was intramuscularly injected in order to relax the cervix; or 2) Warm extender (WE) was deposited in the cervix to modify the cervical muscle dynamics. After the application of these treatments, the success rates achieved with the MpC were 34.2% and 23.8% for VC and WE, respectively. No statistically significant differences were found in the reproductive parameters measured [farrowing (%), litter size and fecundity index] between the use of MpC, or the MpC combined with VC or WE, compared with gilts inseminated by cervical AI (control group). In the second experiment, new catheters based on the anatomical characteristics of gilts (GpC) were used, and the rate of successful pC-AI application were compared (experiment 2a): a) MpC: control; b) GpC1: multi-ring catheter of Ø 16 mm and inner cannula of Ø 3.5 mm; c) GpC2: a multi-ring catheter of Ø with an inner cannula of Ø 2.5 mm. The highest rate of successful cannula penetration was reached in the GpC2 group (60.3%) followed by GpC1 (37.0%) and MpC (19.6%) (p < 0.05). There were no differences in the above mentioned reproductive parameters using the three catheters compared with cervical AI method (control group). Moreover, prior cervical AI did not improve subsequent pC-AI application 24 h later (experiment 2b). In conclusion, Vetrabutin Chlorhydrate, warm extender or the new catheters can be considered as useful tools for improving the success rate of pC-AI technique in gilts.

Reproductive performance and backflow study in cervical and post-cervical artificial insemination in sows.

The present study was developed to evaluate multiparous sow reproductive performance and backflow in post-cervical artificial insemination (post-CAI) using a reduced number of sperm than in cervical artificial insemination (CAI). The experimental groups were divided into sows inseminated by: 1) cervical artificial insemination (CAI): 3×10(9) spermatozoa/80 ml; 2) post-CAI: 1.5×10(9) spermatozoa/40 ml (post-CAI 1); 3) post-CAI using 1×10(9) spermatozoa/26 ml (post-CAI 2). Post-CAI 1 reproductive parameters were similar to those of post-CAI 2 (except for live born litter size which was greater in post-CAI 1) and better than for the CAI group (p<0.01). In a second experiment the backflow volume, number of sperm, and sperm quality in the backflow were studied in the 3 experimental groups. The % of volume and spermatozoa in the backflow was higher in the CAI group (p<0.05) than post-CAI groups (statistically similar between them). Moreover, the quality parameters (motility, progressive motility, viability, chromatin decondensation and morphology) in backflow semen were identical in all three experimental groups, but differed as regards the original insemination dose incubated inside a colostomy bag (sperm quality control group). The present study shows that the use of post-CAI (either post-CAI 1 or 2) in field conditions can be recommended because the efficiency is similar (in the case of post-CAI 2) or higher (post-CAI 1) than when using the traditional method (CAI), representing a reduction cost.

Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA.

Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) protein-coated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 microg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.