Mario Párraga

Role of phospholipase A2 in mammalian sperm-egg fusion: Development of hamster oolemma fusibility by lysophosphatidylcholine

Phospholipase A2 (PLA2), its localization on human sperm and its involvement in sperm-egg interaction, was investigated. Sperm-egg interaction was examined using an in vitro assay of the interaction between human sperm and zona-free or zona-intact hamster egg. PLA2- specific antibodies and/or lysophosphatidylcholine (LPC) were added to the coincubation medium. PLA2 was localized on the anterior tip of the human sperm head by an immunogold silver staining method in light microscopy (IGSS) and TEM. PLA2-specific antibodies inhibited human sperm-zona-free oocyte fusion significantly. LPC treatment allows interspecies fertilization of zona-intact hamster oocytes. PLA2 plays an important role in membrane-fusion events. This statement is supported by the fact that PLA2 is localized in the region where an exocytotic event, such as the acrosome reaction, occurs in the spermatozoon. PLA2-specific antibodies inhibited sperm-egg fusion, but not sperm-oolemma adhesion. LPC may stimulate the fertilizing ability of spermatozoa and induce changes on the zona pellucida and on the oolemma promoting in sperm-egg fusion. Based on these findings, it is suggested that sperm PLA2 and one of its modulators, the LPC, may contribute to membrane-fusion events in mammalian fertilization.

Study of the acrosome reaction and the fertilizing ability of hamster epididymal cauda spermatozoa treated with antibodies against phospholipase A2 and/or lysophosphatidylcholine

The present report describes experiments in vitro that were designed to evaluate the involvement of phospholipase A2 (PLA2) in the acrosome reaction of mammalian sperm and the interaction of gametes. Hamster spermatozoa were incubated in a defined medium (TALP) to induce capacitation and the acrosome reaction. This medium was supplemented with antibodies against porcine pancreatic PLA2 and/or lysophosphatidylcholine (LPC). For in vitro fertilization, spermatozoa and/or oocytes were incubated in TALP medium that contained PLA2-specific antibodies, LPC, or antibodies plus LPC. The antibodies inhibited the acrosome reaction in a dose-dependent manner, without any effect on sperm motility or hyperactivation. These antibodies also inhibited fertilization in vitro. LPC, a product of the reaction catalysed by PLA2, speeds up and synchronizes the acrosome reaction and facilitates penetration of the zona pellucida by spermatozoa, the fusion process and polyspermy. The results of addition of the antibodies plus LPC showed that LPC is able to reverse the inhibitory effects of the antibodies on the acrosome reaction and fertilization. It is possible that endogenous PLA2 plays a role in the final stages of the acrosome reaction and the interaction of gametes, perhaps through one of its reaction products, LPC. The role of LPC might be to stimulate the fertilizing ability of spermatozoa, as well as to induce changes in the zona pellucida and the oolemma that allow sperm-egg fusion. Thus, it seems possible that PLA2 and one of its reaction products might contribute to membrane-fusion events during mammalian fertilization.

Fhx(Foxj2) expression is activated during spermatogenesis and very early in embryonic development

FHX (FOXJ2) is a recently characterized human fork head transcriptional activator that binds DNA with a dual sequence specificity. We have cloned the cDNA for the mouse orthologue Foxj2 and characterized its expression in the gonads and along the early pre-implantation development of the mouse. In the testis, Foxj2 is expressed from pachytene spermatocytes to round spermatids, but not in spermatogonia. In addition to the germ lineage, only Sertoli cells of the testis showed expression of Foxj2. In the ovary, only granulosa cells of the follicles express the factor. Neither mature spermatozoa nor oocytes showed expression of Foxj2. Foxj2 expression is early activated in zygotic development, being detected since as early as 8-cell stage embryos. Both cell layers of the blastocyst: the trophectoderm (TE) and the inner cell mass (ICM), express Foxj2.

XYbp, a novel RING-finger protein, is a component of the XY body of spermatocytes and centrosomes.

RING-finger proteins participate in developmental processes, including gametogenesis. A fetal oocyte cDNA library was used to select genes expressed during male germ-cell differentiation. A novel RING-finger protein, XYbp (XY body protein), participating in mouse spermatogenesis has been identified. This novel gene generates a ubiquitously expressed transcript of 4.2 kb and a testis-specific one of 2.8 kb, processed by an alternative polyadenylation mechanism from a non-canonical polyadenylation signal. Transcription of XYbp is regulated during spermatocyte differentiation. The antiserum raised against the XYbp peptide demonstrated that XYbp is localised mainly in the XY bivalent of spermatocytes (XY body) and in the centrosomes of somatic and germ cells in all phases of the cell cycle. These studies indicate that we have identified a new member of the RING-finger family of proteins associated with the XY meiotic bivalent during spermatogenesis development and with the centrosomes of all cells.

Regulated expression of p14 (cofactor A) during spermatogenesis

The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1. p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route. Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell. This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur. Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues. Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids. The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself. Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers. The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance. As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation.

Ilf2 is regulated during meiosis and associated to transcriptionally active chromatin.

Analysis of gene expression during testis development demonstrated accumulation of Ilf2 mRNA in pachytene spermatocytes. In these cells, the protein was localized in the nucleus, but it was absent from chromatin of the XY pachytene bivalent, in which there is no transcriptional activity. Nucleolar signal is inmmunolocalized in spermatogonia, Sertoli cells and oocytes. By in situ hybridisation, Ilf2 expression is detected in proliferative cells of adult ovary and a defined pattern is also exhibited in different tissues of embryos. The presence of ILF2 in active chromatin is corroborated in NIH3T3 cultured cells after transfection with Ilf2-EGFP constructs.

Differential expression of Ran GTPase during HMBA-induced differentiation in murine erythroleukemia cells

Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA). To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-induced cells by differential hybridization and isolated GTPase Ran as a down-regulated gene. We observed that Ran was expressed in a biphasic mode. Following a decrease in mRNA level during the initial hours of induction, Ran re-expressed at 24-48 h, and gradually declined again. To investigate the role of Ran during MEL differentiation we constructed MEL transfectants capable to express or block Ran mRNA production constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL cell differentiation resulting in a decrease of cell survival in the committed population.