New insights into the pathogenesis of cystic follicles in cattle: Microarray analysis of gene expression in granulosa cells1

Abstract

Ovarian follicular growth and development are regulated by extraovarian and intraovarian factors, which influence granulosa cell proliferation and differentiation. However, the molecular mechanisms that drive follicular growth are not completely understood. Ovarian follicular cysts are one of the most common causes of reproductive failure in dairy cattle. Nevertheless, the primary cause of cyst formation has not been clearly established. A gene expression comparison may aid in elucidating the causes of ovarian cyst disease. Our objective was to identify differentially expressed genes in ovarian granulosa cells between normal dominant and cystic follicles of cattle. Granulosa cells and follicular fluid were isolated from dominant and cystic follicles collected via either ultrasound-guided aspiration from dairy cows (n = 24) or slaughterhouse ovaries from beef cows (n = 23). Hormonal analysis for progesterone, estradiol, and androstenedione in follicular fluid was performed by RIA. Total RNA was extracted and hybridized to 6 Affymetrix GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA). Abundance of mRNA for differentially expressed selected genes was determined through quantitative real-time reverse-transcription PCR. Follicular cysts showed greater (P < 0.05) progesterone, lesser (P < 0.05) estradiol, and no differences (P > 0.10) in androstenedione concentrations compared with noncystic follicles. A total of 163 gene sequences were differentially expressed (P < 0.01), with 19 upregulated and 144 downregulated. From selected target genes, quantitative real-time reverse-transcription PCR confirmed angiogenin, PGE(2) receptor 4, and G-protein coupled receptor 34 genes as upregulated in cystic follicles, and Indian hedgehog protein precursor and secreted frizzled-related protein 4 genes as downregulated in cystic follicles. Further research is required to elucidate the role of these factors in follicular development and cyst formation.

Alteration in localization of steroid hormone receptors and coregulatory proteins in follicles from cows with induced ovarian follicular cysts

“…However, numerous studies have shown that cysts are not simply inactive persistent structures but are in fact the contrary, still evolved, with important metabolic activity and structural changes , Monniaux et al 2008, Ortega et al 2008, Grado-Ahuir et al 2011, Rodríguez et al 2011. Also, other authors have found similar changes in the protein and mRNA expression of ESR2 isoforms in follicular cysts in women with polycystic ovary syndrome compared with normal size-matched follicles in healthy women (Jakimiuk et al 2002), and in prenatal testosterone-treated ovine females characterized by follicular persistence (Ortega et al 2009).…”

Expression and effect of fibroblast growth factor 9 in bovine theca cells

“…The bovine CYP11A1 and FGF9 primer and probe sequences and information are described by Lagaly et al (2008) and Grado-Ahuir et al (2011) respectively. The bovine CYP17A1, STAR, and LHCGR primer and probe sequences are described by Spicer et al (2008).…”

“…Furthermore, the RT-PCR products were run on agarose gels to verify the length and size of the expected target genes, and the same RT-PCR cDNA samples were sequenced to verify the amplified product. Target gene expression was normalized to constitutively expressed 18S rRNA and relative quantity of target gene mRNAs was expressed as 2 KDDCt using the relative comparative threshold cycle method as described previously (Lagaly et al 2008, Grado-Ahuir et al 2011). …”

“…The peptide hormone fibroblast growth factor 9 (FGF9) is downregulated in cystic follicles compared with non-cystic follicles of cattle (Grado-Ahuir et al 2011), indicating that FGF9 may play a role in follicular development and cyst formation. In another microarray study, FGFR2IIIc mRNA, one of the receptors for FGF9 (Ornitz et al 1996), was upregulated by insulin-like growth factor 1 (IGF1) in cultured porcine granulosa cells (GCs; Grado-Ahuir et al 2009), suggesting that FGF9 may also be involved in porcine follicular growth.…”

Treatment of Ovarian Cysts in Dairy Cows with Simultaneous Administration of GnRH and PGF2α has no Clear Advantage Over the Use of GnRH Alone

“…This disorder is a significant cause of reproductive losses with great economic impact on dairy farms. The incidence of COF is high, since in many herds this disorder has been estimated at 10%-30% of the population (10,22,28). Economic losses are caused by an increase in the number of days open, increased culling rate because of infertility, and high treatment costs (8).…”