Korean J Physiol Pharmacol.  2010 Oct;14(5):265-272. 10.4196/kjpp.2010.14.5.265.

Identification of Differentially Expressed Genes in Bovine Follicular Cystic Ovaries

Affiliations
  • 1Department of Physiology, Institute of Health Sciences, Gyeongsang National University School of Medicine, Jinju 660-751, Korea. dawon@gnu.ac.kr
  • 2Animal Genetic Resources Station, National Institute of Animal Science, RDA, Namwon 590-832, Korea.
  • 3Department of Obstetrics and Gynecology, Samsung Changwon Hospital, Sungkyunkwan University School of Medicine, Changwon 630-723, Korea.

Abstract

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and > or =25 mm). These follicles had granulosa cell layer and theca interna and the hormone 17beta-estradiol (E2)/ progesterone (P4) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using GeneFishing(TM) DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.

Keyword

Gene expression; Estrogens; Follicular cyst

MeSH Terms

Blotting, Western
Databases, Nucleic Acid
Discrimination (Psychology)
Down-Regulation
Estrogens
Female
Follicular Cyst
Gene Expression
Granulosa Cells
Microtubule-Associated Proteins
Ovarian Diseases
Ovary
Pathologic Processes
Polymerase Chain Reaction
Progesterone
Ribosomal Proteins
RNA
Theca Cells
Up-Regulation
Estrogens
Microtubule-Associated Proteins
Progesterone
RNA
Ribosomal Proteins

Figure

  • Fig. 1. The structure of annealing control primer (ACP). The ACP is composed of three sequence regions. (A) Target core sequence (3′- end targeting portion; 10 nts) containing a hybridizing sequence substantially complementary to a site on a template nucleic acid (B) Universal sequence (5′- end portion; 22 nts) (C) Regulator sequence (linker portion separated 3′- end portion and 5′- end portion; 5 nts).

  • Fig. 2. Microphotographs of sections of bovine ovary (A) HE staining of normal ovary section and (B) follicular cystic ovary. Normal ovaries (a, b, and c) comprise 8 to 12 diverse granulosa cell layers. GC, and TI represent granulosa cell and theca interna, respectively. Bars =100 μm. (C) TUNEL staining of bovine ovary section. Tissue sections showing averaged value were represented. A tissue section isolated from normal ovary was used for negative control (left panel), and normal and cystic ovary sections containing follicles were shown in middle and right panels, respectively. The bar graph shows normalized fluorescence intensities for normal and cystic ovaries. The fluorescence intensity of normal and cystic ovaries was normalized to that of negative control. Each bar represents mean±SD of five experiments.

  • Fig. 3. Differentially expressed genes (DEGs) of normal and follicular cystic follicles (FCFs) in Korean cattle. (A) Photographs of gels containing DEGs from bovine follicles. Using 20 ACPs (GP1 to GP20), two DEGs were identified in FCFs. Arrows indicate DEG bands (numbers 1 and 2). These DEG bands were excised from the gel for further cloning and sequencing. GP, N, and C represent general primer, normal ovary, and follicular cystic ovary, respectively. (B) Expression level of RPL-15 and MAP1B derived from normal follicles and FCFs. The mRNA expression level was analyzed using an ACP-based GeneFishing polymerase chain reaction (PCR). Each bar represents mean ±SD of three experiments. The asterisks indicate a significant difference from the corresponding control value obtained for normal follicles (p<0.05). (C) RT-PCR products for RPL15 and MAP1B derived from bovine ovaries. Bovine follicles showed 251-bp, 302-bp, and 386-bp bands for GAPDH, RPL-15, and MAP1B, which correspond to the expected lengths. The first lane shows a 1-kb DNA ladder. The bar graph shows normalized mRNA levels for RPL15 and MAP1B. The mRNA expression of RPL-15 and MAP1B was normalized to that of GAPDH. Each bar represents mean±SD of five experiments. The asterisks indicate a significant difference from the corresponding control value obtained for normal ovaries (p<0.05).

  • Fig. 4. Western blot analysis of RPL-15 and MAP1B in bovine follicular cystic follicles. (A) Upregulation of RPL-15 protein in bovine follicular cystic follicles (FCFs). (B) The bar graph shows normalized protein levels of RPL-15 in the bovine FCFs. The expression levels were normalized to β-actin. (C) Down-regulation of MAP1B in FCFs. PC12 cells were used as a positive control. Equal amounts (100 μg) of total protein were loaded in each lane. Molecular weight is indicated on the left side of the blot. (D) The bar graph shows normalized protein levels of MAP1B against β-actin in the bovine FCFs. Each bar represents mean±SD of five experiments. The asterisks indicate a significant difference from the corresponding control value obtained for normal follicles (p<0.05).


Reference

References

1. Peter AT. An update on cystic ovarian degeneration in cattle. Reprod Domest Anim. 2004; 39:1–7.
Article
2. Hamilton SA, Garverick HA, Keisler DH, Xu ZZ, Loos K, Youngquist RS, Salfen BE. Characterization of ovarian follicular cysts and associated endocrine profiles in dairy cows. Biol Reprod. 1995; 53:890–898.
3. Hauptmann S, Denkert C, Koch I, Petersen S, Schluns K, Reles A, Dietel M, Petersen I. Genetic alterations in epithelial ovarian tumors analyzed by comparative genomic hybridization. Hum Pathol. 2002; 33:632–641.
Article
4. Osterberg L, Akeson M, Levan K, Partheen K, Zetterqvist BM, Brannstrom M, Horvath G. Genetic alterations of serous borderline tumors of the ovary compared to stage I serous ovarian carcinomas. Cancer Genet Cytogenet. 2006; 167:103–108.
5. Lingenfelter BM, Dailey RA, Inskeep EK, Vernon MW, Poole DH, Rhinehart JD, Yao J. Microarray analysis of gene expression in granulosal cells from persistent follicles in cattle. Anim Reprod Sci. 2008; 104:405–413.
Article
6. Hwang IT, Kim YJ, Kim SH, Kwak CI, Gu YY, Chun JY. Annealing control primer system for improving specificity of PCR amplification. Biotechniques. 2003; 35:1180–1184.
Article
7. Kim YJ, Kwak CI, Gu YY, Hwang IT, Chun JY. Annealing control primer system for identification of differentially expressed genes on agarose gels. Biotechniques. 2004; 36:424–426. 428, 430 passim.
Article
8. Kesler DJ, Elmore RG, Brown EM, Garverick HA. Gonadotropin releasing hormone treatment of dairy cows with ovarian cysts. I. Gross ovarian morphology and endocrinology. Theriogenology. 1981; 16:207–217.
Article
9. Isobe N, Yoshimura Y. Deficient proliferation and apoptosis in the granulosa and theca interna cells of the bovine cystic follicle. J Reprod Dev. 2007; 53:1119–1124.
Article
10. Isobe N, Yoshimura Y. Localization of apoptotic cells in the cystic ovarian follicles of cows: a DNA-end labeling histochemical study. Theriogenology. 2000; 53:897–904.
Article
11. Calder MD, Manikkam M, Salfen BE, Youngquist RS, Lubahn DB, Lamberson WR, Garverick HA. Dominant bovine ovarian follicular cysts express increased levels of messenger RNAs for luteinizing hormone receptor and 3 beta-hydroxysteroid dehydrogenase delta(4), delta(5) isomerase compared to normal dominant follicles. Biol Reprod. 2001; 65:471–476.
12. Isobe N, Kitabayashi M, Yoshimura Y. Microvascular distribution and vascular endothelial growth factor expression in bovine cystic follicles. Domest Anim Endocrinol. 2005; 29:634–645.
Article
13. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson G, Enmark E, Pettersson K, Warner M, Gustafsson JA. Mechanisms of estrogen action. Physiol Rev. 2001; 81:1535–1565.
Article
14. Matthews J, Gustafsson JA. Estrogen signaling: a subtle balance between ER alpha and ER beta. Mol Interv. 2003; 3:281–292.
15. Cochrane AW, Deeley RG. Estrogen-dependent modification of ribosomal proteins. Effects of estrogen withdrawal on the distribution of constitutive and hormonally regulated mRNAs. J Biol Chem. 1984; 259:15408–15413.
Article
16. Sowers JR. Estrogen-inducible cytoskeletal linker protein ezrin interaction with the low-density lipoprotein receptor. Endocrinology. 2004; 145:3074.
Article
17. Malayer JR, Cheng J, Woods VM. Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1. Biol Reprod. 1999; 60:1204–1210.
18. Wang Q, Yang C, Zhou J, Wang X, Wu M, Liu Z. Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer. Gene. 2001; 263:205–209.
Article
19. Wang H, Zhao LN, Li KZ, Ling R, Li XJ, Wang L. Overexpression of ribosomal protein L15 is associated with cell proliferation in gastric cancer. BMC Cancer. 2006; 6:91.
Article
20. Chen FW, Ioannou YA. Ribosomal proteins in cell proliferation and apoptosis. Int Rev Immunol. 1999; 18:429–448.
Article
21. Wool IG. Extraribosomal functions of ribosomal proteins. Trends Biochem Sci. 1996; 21:164–165.
Article
22. Caetano AR, Johnson RK, Pomp D. Generation and sequence characterization of a normalized cDNA library from swine ovarian follicles. Mamm Genome. 2003; 14:65–70.
Article
23. Yao J, Ren X, Irel JJ, Coussens PM, Smith TP, Smith GW. Generation of a bovine oocyte cDNA library and microarray: resources for identification of genes important for follicular development and early embryogenesis. Physiol Genomics. 2004; 19:84–92.
Article
24. Bettegowda A, Patel OV, Ireland JJ, Smith GW. Quantitative analysis of messenger RNA abundance for ribosomal protein L-15, cyclophilin-A, phosphoglycerokinase, beta-glucuronidase, glyceraldehyde 3-phosphate dehydrogenase, beta-actin, and histone H2A during bovine oocyte maturation and early embryogenesis in vitro. Mol Reprod Dev. 2006; 73:267–278.
25. Vallee RB, Davis SE. Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1). Proc Natl Acad Sci U S A. 1983; 80:1342–1346.
Article
26. Bloom GS, Luca FC, Vallee RB. Microtubule-associated protein 1B: identification of a major component of the neuronal cytoskeleton. Proc Natl Acad Sci U S A. 1985; 82:5404–5408.
Article
27. Binder LI, Frankfurter A, Kim H, Caceres A, Payne MR, Rebhun LI. Heterogeneity of microtubule-associated protein 2 during rat brain development. Proc Natl Acad Sci U S A. 1984; 81:5613–5617.
Article
28. Cueille N, Blanc CT, Riederer IM, Riederer BM. Microtubule-associated protein 1B binds glyceraldehyde-3-phosphate dehydrogenase. J Proteome Res. 2007; 6:2640–2647.
Article
29. Cooley L, Theurkauf WE. Cytoskeletal functions during Drosophila oogenesis. Science. 1994; 266:590–596.
30. Uchida Y. Overexpression of full-length but not N-terminal truncated isoform of microtubule-associated protein (MAP) 1B accelerates apoptosis of cultured cortical neurons. J Biol Chem. 2003; 278:366–371.
Article
31. Shah RD, Anderson KL, Rapoport M, Ferreira A. Estrogen-induced changes in the microtubular system correlate with a decreased susceptibility of aging neurons to beta amyloid neurotoxicity. Mol Cell Neurosci. 2003; 24:503–516.
Article
32. Lee ST, Han HJ, Oh SJ, Lee EJ, Han JY, Lim JM. Influence of ovarian hyperstimulation and ovulation induction on the cytoskeletal dynamics and developmental competence of oocytes. Mol Reprod Dev. 2006; 73:1022–1033.
Article
33. Tanner SL, Franzen R, Jaffe H, Quarles RH. Evidence for expression of some microtubule-associated protein 1B in neurons as a plasma membrane glycoprotein. J Neurochem. 2000; 75:553–562.
Article
34. Way AL. Isolation and culture of bovine oviductal epithelial cells for use in the anatomy and physiology laboratory and undergraduate research. Adv Physiol Educ. 2006; 30:237–241.
Article
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