SUMMARYThere have been several reports of an increased risk of genomic imprinting disorders associated with assisted reproductive technology (ART). However, the connection between imprinting defects and ART is tenuous. In this review, this putative association is investigated in detail, with emphasis on particular steps of the ART process, and which of these may be prone to induction of imprinting errors due to synchrony with major imprinting events during gametogenesis, fertilization, and early embryonic development. While contributions from in vitro manipulation of gametes and embryos cannot be ruled out, it appears that superovulation and/or the condition of infertility, itself, may be largely responsible for the increased risk of genomic imprinting disorders observed with ART births. However, two significant shortcomings of all of these studies preclude rigorous exploration of this issue: the lack of large, longitudinal studies on specific cohorts of ART-conceived children, and questions surrounding the primary generation of expression and epigenetic data from oocytes and embryos. Future directions for study are proposed, along with a preview of what may be in store as the art of ART advances.

Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE‐PCR approach, or any other PCR‐based method, requires many primer sets and amplification reactions to scan the entire protein‐coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family‐specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient‐specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication. Hum Mutat 12:393–402, 1998. © 1998 Wiley‐Liss, Inc.

Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, laborintensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.

We have begun to isolate gene sequences that are specifically expressed in hematopoietic stem cells (HSCs). There are at least three fundamental requirements for the isolation of HSC-specific transcripts. First, highly enriched populations of HSCs, and an HSC-depleted cell population for comparison must be isolated. Secondly, the gene isolation procedures must be adapted to accommodate the small amounts of RNA obtained from purified HSCs. Finally, a defined screening strategy must be developed to focus on sequences to be examined in more detail. In this report, we describe the characterization of populations of HSCs that are highly enriched (Lin- c-kitHI) or depleted (Lin- c-kitNEG) of HSCs. We compared two methods for gene isolation, differential display polymerase chain reaction (DD-PCR) and subtractive hybridization (SH), and found that the latter was more powerful and efficient in our hands. Lastly we describe the strategy that we have developed to screen clones for further study.

This study was undertaken (1) to devise a method of inducing multiple follicular development and subsequent ovulation in the Djungarian or Siberian hamster (Phodopus sungorus) and (2) to assess the quality of ovulated oocytes collected from PMSG/hCG treated animals in comparison to naturally ovulating animals. Hamsters (4-5 weeks; n = 70) received 5 IU PMSG followed 50-52 hr later by 10 IU hCG. Ovulated oocytes were collected 14-20 hr after hCG injection. Ovulated oocytes were flushed from oviducts of cycling animals (7-12 weeks; n = 30) exhibiting two consecutive estrous cycles. Oocytes were fixed and subjected to triple fluorescence immunostaining using anti-tubulin antibodies, fluorescein phalloidin, and Hoechst 33258. The mean number of ovulated oocytes collected from cycling animals was 4.8 +/- 0.4 (range 1-7). Ovulation occurred in 73% of the PMSG/hCG-stimulated animals. The mean number of oocytes ovulated from stimulated animals was 9.2 +/- 0.8 (range 0-22). The ovaries of animals that did not ovulate or that ovulated few oocytes did respond to PMSG, as indicated by the presence of multiple follicular development and pre-ovulatory stigmata. There was no evidence of a polar body in ovulated oocytes collected from PMSG/hCG-treated or cycling animals, indicating that oocytes were arrested in meiosis I. In the majority (80%) of ovulated oocytes from PMSG/hCG-treated and cycling animals, cortically placed chromosomes were aligned on a metaphase plate equidistant from a bipolar spindle. Sparse f-actin staining was observed in the region of the ooplasm surrounding the chromosomes.(ABSTRACT TRUNCATED AT 250 WORDS)