D.P.L. Green

Gene expression in the mouse preimplantation embryo

Mouse preimplantation development represents a tightly controlled programme of gene expression and cell division, which starts with the fertilized egg and ends with implantation of the blastocyst approximately 4.5 days later. Spatial and temporal differences in gene expression underpin establishment of axes at the two-cell stage and development of the trophectoderm and inner cell mass after embryo compaction at the eight-cell stage. Approximately 15 700 mouse genes expressed during preimplantation development have been identified from cDNA sequences deposited in the UniGene database of the National Institutes of Health. This inventory of preimplantation genes is the starting point for identifying signalling modules that function in preimplantation development.

Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates

Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.

Gene expression profiling of human GV oocytes: an analysis of a profile obtained by serial analysis of gene expression (SAGE)

A gene expression profile of the human GV oocyte has recently been established by Serial Analysis of Gene Expression (SAGE). A significant number of the genes identified in this profile had not previously been associated with mammalian oocytes. We sought to confirm gene matches by RT-PCR amplification of candidate transcripts using mouse eggs. Attention focused on receptors, proteins involved in apoptosis, and cytoskeletal proteins. Two receptors found in the human catalogue, CCR6 and PAR3, were not found in mouse eggs, whereas myosin light chain, LLGL, beta-actin, 5HT receptor, bad, bak, DFF45, and Caspase homologue (cash) were. Individual SAGEtags can match more than one gene and, in some cases, more than ten. Examination of transcript sequences that generate multiple gene assignments identified a common denominator of short interspersed elements or Alu sequences. For reasons which are, as yet, unclear, the human GV oocyte SAGE catalogue contains relatively high abundances of SAGEtags in Alu sequences. This may reflect normal expression of Alu-containing genes in eggs or upregulated expression of Alu elements following stress. The degeneracy of gene matches in SAGE generated by Alu sequences makes independent confirmation of candidate genes essential.

Using expressed sequence tag databases to identify ovarian genes of interest

GenBank contains 4879 expressed sequence tags (EST) derived from four non-normalized human ovarian cDNA libraries. Of these EST, 2646 are contributors to UniGene clusters and have UniGene numbers. The EST map to 1206 distinct UniGenes. A gene expression profile was established for the human ovary by identifying the abundance of each UniGene cluster and its corresponding annotation. The most highly expressed transcripts were for proteins associated with protein synthesis (ribosomal proteins, elongation factors, thymosins, etc.). However, there are also transcripts for genes of unknown function that are ovary-specific. This ovarian gene expression profile provides useful data for the design of DNA microarrays targeted at ovarian function and highlights novel sequences that warrant further investigation.