The objectives of this study were to evaluate: (1) the nuclear maturation, (2) the intracellular glutathione (GSH) content, (3) the normality of fertilization and (4) full development after transplantation of embryos derived from porcine oocytes pre-cultured with 50 μmol/l roscovitine (an inhibitor of p34cdc2/cyclin B kinase) for 22 h. After treatment with roscovitine, the nuclear configuration of oocytes (Hoechst staining) was comparable with those examined just after collection: the majority of oocytes were arrested at the germinal vesicle (GV) 1 stage (63.2%). Roscovitine-treated oocytes progressed through meiosis to the metaphase II stage in a conventional step-wise in vitro maturation (IVM) program for 44 h in a proportion similar to control ones (>85.0%). When roscovitine-treated oocytes and non-treated oocytes were matured for 44 h and then co-cultured with fresh spermatozoa for 18 h, no differences were observed in oocyte penetrability, proportion of monospermic penetration and male pronuclear formation (>87%). Roscovitine increased the GSH synthesis in oocytes at 22 h, whereas, after 44 h, roscovitine-treated oocytes had similar amounts of GSH to non-treated oocytes. Finally, surgical transfer of zygotes at 22–24 h post-insemination, derived from roscovitine-treated oocytes, resulted in one pregnancy with 12 piglets born; control non-treated zygotes resulted in one pregnancy and 10 piglets born. The full-term developmental ability of mammalian oocytes pre-cultured with roscovitine prior to IVM is thereby demonstrated. This validation is important before the introduction of roscovitine into routine procedures.
Pilar Coy, Raquel Romar, Salvador Ruiz, Sebastián Cánovas, Joaquín Gadea, Francisco García Vázquez and Carmen Matás
Maria Jose Ruiz Magaña, Jose Maria Puerta, Rocio Martínez-Aguilar, Tatiana Llorca, Osmany Blanco, Raquel Muñoz-Fernández, Enrique G Olivares and Carmen Ruiz-Ruiz
Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context – that is, non-pregnancy vs pregnancy – of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.
Francisco A García-Vázquez, Salvador Ruiz, Carmen Matás, M José Izquierdo-Rico, Luis A Grullón, Aitor De Ondiz, Luis Vieira, Karen Avilés-López, Alfonso Gutiérrez-Adán and Joaquín Gadea
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 μg/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.