Subtractive hybridization used to identify mRNA associated with the maturation of bovine oocytes

• Published in: Molecular reproduction and development Vol. 57; no. 2; pp. 167 - 175
• Main Authors: Robert, Claude, Barnes, Frank L., Hue, Isabelle, Sirard, Marc-André
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01-10-2000; Wiley-Liss; Wiley
• Subjects: Animals; Biological and medical sciences; Cattle; Cyclin B - genetics; Cyclin B1; differential display; DNA, Complementary - genetics; expression; Female; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; Life Sciences; Mammalian female genital system; Morphology. Physiology; mRNA; Nucleic Acid Hybridization - methods; Oocytes - growth & development; Oocytes - metabolism; Polymerase Chain Reaction; Reproductive Biology; RNA, Messenger - genetics; RNA, Messenger - metabolism; technique; Vertebrates: reproduction; Molecular hybridization; Bovine; Germinal cell; Gene expression; Polymerase chain reaction; Ovary; Vertebrata; Mammalia; Messenger RNA; Animal; Female genital system; Artiodactyla; Ovarian follicle; Ungulata; Oocyte; Oocyte maturation; NORTHERN BLOT; ADNC; ARM MESSAGER
• ISSN: 1040-452X; 1098-2795
• DOI: 10.1002/1098-2795(200010)57:2<167::AID-MRD8>3.0.CO;2-P

The main objective of this project is to identify mRNA associated with oocyte maturation and embryonic developmental competency. The knowledge of genes and their accumulated mRNA is essential to better understand the mechanisms involved in the oocyte maturation and the survival of the in vitro produced embryo. We used bovine slaughterhouse‐recovered ovaries and collected the oocytes from two follicle size categories: <2 mm and 3–5 mm. The mRNA content of oocytes from follicles 3–5 mm where considered to be more competent when compared to the content of oocytes from follicles <2 mm. In this report we compare two different technical approaches both involving PCR to compare the mRNA pools of the oocytes. In the first approach we performed the differential display (DDRT) technique to amplify and display side by side the cDNAs of groups of 10 denuded oocytes. From this approach, we isolated 28 different bands. After analysis, three of those bands had strong homology with known genes. In the second approach pools of 50 denuded oocytes were submitted to suppressive subtraction hybridization (SSH). We identified several known genes like cyclin B1, splicing factor ccl.4, cytochrome c oxidase, and mineralocorticoid receptor while numerous other clones remain unidentified. The cyclin B1 clone was used as a probe to evaluate its follicular size specificity on virtual Northern blot. The PCR basis of these techniques allows comparison of mRNA from tissues of low abundance such as oocytes. In this study the SSH resulted in longer clones than DDRT and showed high specificity. Mol. Reprod. Dev. 57:167–175, 2000. © 2000 Wiley‐Liss, Inc.