Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis

Abstract

The oocyte is a key regulator of ovarian folliculogenesis and early embryonic development. However, the composition of the oocyte transcriptome and identities and functions of key oocyte-specific genes involved in the above processes are relatively unknown. Using a PCR-based cDNA amplification method (SMART technology), we constructed a bovine oocyte cDNA library. Analysis of 230 expressed sequence tags (ESTs) from this library identified 102 unique sequences. Although some correspond to housekeeping genes (e.g., ribosomal protein L15) and some represent genes previously known to be expressed in oocytes and other tissues, most encode for genes whose expression in mammalian oocytes has not been reported previously (e.g., cocaine- and amphetamine-regulated transcript) or genes of unknown function. Sixteen did not show significant sequence similarity to any entries in the GenBank database and were classified as novel. Using over 2,000 unsequenced, randomly selected cDNA clones from the library, we constructed an oocyte microarray and performed experiments to identify genes preferentially expressed in fetal ovary (an enriched source of oocytes) relative to somatic tissues. Eleven clones were identified by microarray analysis with consistently higher expression in fetal ovaries (collected from animals at days 210-260 of gestation) compared with spleen and liver. DNA sequence analysis of these clones revealed that two correspond to JY-1, a novel bovine oocyte-specific gene. The remaining nine clones represent five identified genes and one additional completely novel gene. Increased abundance of mRNA in fetal ovary for five of the six genes identified was confirmed by real-time PCR. Results demonstrate the potential utility of these unique resources for identification of oocyte-expressed genes potentially important for reproductive function.

Gene expression profiling of bovine preovulatory follicles: gonadotropin surge and prostanoid-dependent up-regulation of genes potentially linked to the ovulatory process

“…SSC and deionized water, dried by brief centrifugation and further cleaned with compressed air and scanned using GeneTAC LSIV microarray scanner and GeneTAC LS software (Genomic solutions). GeneTAC Integrator 4.0 software was used to process array images, align spots, integrate robot-spotting files with the microarray image, and to export spot intensity data in a Microsoft Excel format as described previously (Suchyta et al 2003, Yao et al 2004.…”

Microarray gene expression analysis in atrophying rainbow trout muscle: a unique nonmammalian muscle degradation model

“…Fluorophors (Cy3 and Cy5) were randomly assigned to RNA from each of the atrophying and nonatrophying muscles to limit the dye effect. cDNA labeling and microarray hybridization procedures were essentially as described (75). In brief, 0.8 g of mRNA from each trout muscle were used as a template in reverse transcription reactions incorporating amino-allyl dUTP into the cDNA using oligo-d (T) primer and Superscript II reverse transcriptase (Invitrogen).…”

JY-1 , an oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle

“…The advent of oocyte genomics and EST sequencing projects have led to a dramatic increase in our understanding about the identities and functions of oocytespecific genes in female reproduction (2,3). However, inherent species-specific differences exist in the ovulation quota, follicular waves, duration of the ovarian cycle, and number of embryonic cell cycles required for embryonic genome activation (4) between the traditional animal model (polyovulatory mouse) versus monoovulatory species such as cattle and primates, including humans.…”

“…Thus, comparative genomics approaches coupled to functional studies in nontraditional model systems are needed to address dissimilarities in transcriptome composition between model organisms and provide information on existence of genes or gene families that may play important regulatory roles in fertility in nonmurine models, including the human. With this goal in mind, we previously constructed a bovine oocyte cDNA library and sequenced a number of ESTs (2). A highly abundant transcript (designated as JY-1) was identified and selected for further analysis, because it is entirely novel despite 7.95 million human, 4.74 million mouse, and Ϸ1.31 million bovine EST sequences in GenBank and because its expression is ovary specific.…”