Maternal Determinants and the Polarised Marsupial Oocyte.

Abstract

Cleavage is highly stylised in dasyurid marsupials that have highly polarized oocytes and zygotes. The first three cleavage divisions are meridional along the axis of polarity and the cells appear identical except for the order of cell division, in which the first cell to divide at each cleavage division lies on one side of the conceptus and the last cell to divide lies opposite it. The fourth division is latitudinal and separates the conceptus into 8 smaller pluriblast cells (equivalent to the inner cell mass) and 8 larger trophoblast cells. The axis between 1st and last dividing cells at each division is maintained in both pluriblast and trophoblast cells. No morula is formed, but instead the cells from early cleavage adhere to the zona pellucida and then to each other from the 8-cell stage so that they form a unilaminar blastocyst, which is completed at about the 32- cell stage. Cleavage in other marsupials such as the possum or the opposum is slightly less stylized, in that from the 4 -cell stage some cells begin to divide latitudinally, but the end result is the same with about half the cells at the 16-cell stage being pluriblast and half trophoblast. Such a specific cleavage pattern suggests that determinants leading to pluriblast vs trophoblast might be differentially allocated at the 16-cell stage. Certainly the vesicular products which are mainly located in the pole opposite the pronuclei end up being progressively restricted to the trophoblast cells and continue to be discharged into the cleavage cavity as they contribute to the blastocoel. We have identified one vesicle-associated protein, VAP1, which is produced during oogenesis from early primary oocyte stages onwards. VAP1 is a cystatin-like protein, which acts as a cysteine proteinase inhibitor and probably assists in stabilizing the extracellular matrix of the blastocoel. Immunization with VAP1 in possums results in accumulations of degraded lipid bodies which partially replace vesicles in oocytes, disruption of normal polarity in ovulated oocytes and zygotes and failure of normal development. In addition, in a recent cDNA RDA study we isolated ~180 clones of which 70 have been tested and 49 have been identified as dunnart ovary-specific, using Southern dot blot hybridization. On investigation of 20 randomly selected clones, 5 dunnart ovary-specific genes, which included ZP-2, c-mos and 3 novel genes as determined by comparing expression in several somatic, as well as gonadal and brain dunnart tissues by RT-PCR, have been identified. Most importantly, the 3 novel gene transcripts are present during stages of early development. This strongly suggests that some of these transcripts may be maternal determinants akin to those for the frog and chick, and play a role in patterning in the marsupial embryo. The patterns displayed by the marsupial embryo and the late onset of the zygote genome activation at the 16- cell stage, suggest that any determinants found will be involved in establishing the pluriblast/trophoblast lineages and the embryonic-abembryonic axis, and setting up a putative anterior-posterior axis in both pluriblast and trophoblast. The research was supported by the New Zealand Foundation for Research Science and Technology and the University of Melbourne.