238 ESTROGEN RECEPTOR ALPHA AND PROGESTERONE RECEPTOR EXPRESSION FROM REPRODUCTIVE TISSUE AND IN VITRO PRODUCED EMBRYOS OF THE DOMESTIC CAT

Abstract

The in vitro production of cat embryos has been reported by several laboratories, and kittens have been born after transfer of embryos derived by IVF, ICSI, and NT. However, evidence accumulating in other species indicates that in vitro-derived embryos exhibit altered gene expression of developmentally important genes. Because the domestic cat genome is not well defined, the lack of primer sequence information poses a challenge for gene expression profiling. Estrogen, in addition to its essential role in the development and function of the female reproductive tract, is important for maturation of the oocyte. The aim of this preliminary study was to evaluate the expression profile of estrogen receptor alpha (ERα) and progesterone receptor (PR) in domestic cat female reproductive tissue and in vitro-produced (IVP) embryos. Embryos were produced in vitro as described by Gomez et al. (2003 Theriogenology 60, 239–251). mRNA was isolated from pools (n = 35–50) of IVP domestic cat embryos at the morula and blastocyst stages (Days 7 and 8) using a modified protocol of the PolyAT Tract System (Promega, Madison, WI, USA). Uterus and ovaries were collected from hystorectomized cats and total RNA was isolated from these estrogen-targeted organs (RNeasy standard protocol, QIAGEN, Valencia, CA, USA). All RNA was reverse-transcribed and subjected to real-time PCR to evaluate expression of ERα and PR. The housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as a control for expression in all tissues. Using primers designed to amplify human sequences or multispecies primers, we successfully amplified all three genes from as little as ∼100 ng of ovarian or uterine mRNA. We were also able to detect ERα and GAPDH from pools of IVP embryos. Transcripts were cloned and confirmed by sequencing to be homologous to known sequences of the respective genes in a variety of species, including human, mouse, and pig. A 234-bp transcript of ERα (accession #AY349164;; GenBank) corresponding to exons 5 through 7 of the human ERα gene (hormone-binding domain) was identified along with a 110-bp sequence of PR and 98-bp sequence of GAPDH. To our knowledge, this is the first description of ERα or PR cloning in the domestic cat. In summary, we have demonstrated that highly sensitive real-time PCR is effective for the assessment of gene expression in cat ovarian and uterine tissue, as well as in embryos, the latter, of which, are known to have a low transcript copy number. Furthermore, these experiments provide the basis for future studies on the effect of exogenous estrogen on gene expression in cat IVM oocytes and IVP embryos.