Follicular growth rates were studied in 5 Hereford-Holstein cross heifers on Day 14 of the oestrous cycle. The granulosa cell mitotic index (MI) was measured in non-atretic antral follicles of various diameters (0.13-8.57 mm) from Bouin-fixed ovaries collected before (199, control) and 2 h after colchicine treatment (189, treated). In control ovaries, follicles of 0.68-1.52 mm had a higher MI than those of other size classes (P less than 0.05). In colchicine-treated ovaries, the MI of follicles ranging from 0.68 to 8.57 mm increased more than that of other sized follicles, so that the mitotic time was shorter (0.78 h vs 1.32 h) in medium and large sized follicles (0.68-8.57 mm) than in smaller follicles (0.13-0.67 mm). Calculations based on the number of granulosa cells in follicles of various classes and from the time required to double the number of cells within a follicle indicate that a follicle takes 27 days to grow from 0.13 to 0.67 mm, 6.8 days from 0.68 to 3.67 mm and 7.8 days from 3.68 to 8.56 mm, indicating that growth rates varied with the size of the follicle. A period equivalent to 2 oestrous cycles would therefore be required for a follicle to grow through the antral phase, i.e. from 0.13 mm to preovulatory size. Increased MI, decreased mitotic time and increased atresia found in follicles larger than 0.68 mm could indicate a change in the follicular metabolism during its maturation.

The objective was to analyze gene expression in bovine granulosa cells of the dominant follicle by mRNA differential display. Total RNA was extracted from granulosa cells of

Molecular determinants and mechanisms involved in ovarian follicular growth, ovulation, and luteinization are not well understood. The objective of this study was to identify genes expressed in bovine granulosa cells (GC) of dominant follicles (DF) and downregulated after hCG-induced ovulation, using the suppression subtractive hybridization (SSH). GC were collected from DF at Day 5 of the estrous cycle and from ovulatory follicles (OF) obtained 23 h following injection of hCG. A subtracted cDNA library (DF-OF) was generated and screened using unsubtracted (DF, OF) and subtracted (DF-OF, OF-DF) cDNAs as complex (32)P-probes. A total of 32 nonredundant cDNAs were identified: 23 cDNAs matched with sequences of known biological function and 9 cDNAs with complete or partial sequences of undefined biological function. Detection of genes known to be downregulated during the periovulatory period in the bovine species, such as CPD, CYP11A1, CYP19A1, FSHR, LRP8/ ApoER2, and SERPINE2, validated the physiological model and analytical techniques used. For a subset of genes, such as ARFGAP3, CYP11A1, CYP19A1, FSHR, FST, GJA1, IDH3, INHBA, LHCGR, LHCGR lacking exon 10, PRC1, PRG1, RPA2, SCD, and TRIB2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction from follicles obtained at different developmental stages. Results confirmed a downregulation of the respective mRNAs in GC of OF compared with that of DF. We conclude that we have identified novel genes that are downregulated by hCG in bovine GC of DF during the periovulatory period, which may contribute to follicular growth, ovulation, and/or luteinization.

To elucidate the molecular mechanisms involved in the delayed induction of PGHS-2 in species with a long ovulatory process, a 1.6-kilobase fragment of the bovine PGHS-2 promoter was isolated, and its activity was characterized in primary cultures of bovine granulosa cells. Promoter activity assays performed with a series of deletion mutants revealed that the promoter region from ؊149 to ؊2 (؉1 ‫؍‬ transcription start site) confers full-length promoter activity in response to forskolin (10 M). Four consensus cis-elements were identified within this region, including an E-box, ATF/CRE, C/EBP, and AP2 site. Site-directed mutagenesis showed that the Ebox was required for PGHS-2 promoter activity, that disruption of the C/EBP element decreased forskolin inducible activity by 29%, whereas point mutation within the ATF/CRE and AP2 element had no inhibitory effect. Electrophoretic mobility shift assays (EMSAs) performed with the ؊149/؊2 fragment and granulosa cell nuclear extracts obtained before (0 h) and after (18 and 20 h) human chorionic gonadotropin (hCG) revealed the regulation of multiple DNA-protein complexes. The 0-h extract generated four complexes at the E-box, whereas only one complex was produced at this site with the 18-h extract. Supershift EMSAs identified that upstream stimulatory factor-1 and -2 (USF-1 and -2) were part of these complexes. Interestingly, the presence of the amino-terminal truncated USF-2, which lacks the transcription activation domain, was detected in the 0-h extract, but not in extracts prepared posthCG. Supershift EMSAs also indicated high levels of C/EBP␤ binding to its cis-element in the 0-h extract, which contrasts with results previously reported in rats. Thus, high levels of amino-terminal truncated USF-2 and C/EBP␤ in bovine granulosa cells prior to hCG treatment could repress gene expression, and be involved in the delayed induction of PGHS-2 in species with a long ovulatory process.

BackgroundAmong the most harmful of all genetic abnormalities that appear in colorectal cancer (CRC) development are mutations of KRAS and its downstream effector BRAF as they result in abnormal extracellular signal-related kinase (ERK) signaling. In a previous report, we had shown that expression of a constitutive active mutant of MEK1 (caMEK) in normal rat intestinal epithelial cells (IECs) induced morphological transformation associated with epithelial to mesenchymal transition, growth in soft agar, invasion and metastases in nude mice. Results from microarrays comparing control to caMEK-expressing IECs identified the gene encoding for serpinE2, a serine protease inhibitor, as a potential target of activated MEK1.Results1- RT-PCR and western blot analyses confirmed the strong up-regulation of serpinE2 expression and secretion by IECs expressing oncogenic MEK, Ras or BRAF. 2- Interestingly, serpinE2 mRNA and protein were also markedly enhanced in human CRC cells exhibiting mutation in KRAS and BRAF. 3- RNAi directed against serpinE2 in caMEK-transformed rat IECs or in human CRC cell lines HCT116 and LoVo markedly decreased foci formation, anchorage-independent growth in soft agarose, cell migration and tumor formation in nude mice. 4- Treatment of CRC cell lines with U0126 markedly reduced serpinE2 mRNA levels, indicating that expression of serpinE2 is likely dependent of ERK activity. 5- Finally, Q-PCR analyses demonstrated that mRNA levels of serpinE2 were markedly increased in human adenomas in comparison to healthy adjacent tissues and in colorectal tumors, regardless of tumor stage and grade.ConclusionsOur data indicate that serpinE2 is up-regulated by oncogenic activation of Ras, BRAF and MEK1 and contributes to pro-neoplastic actions of ERK signaling in intestinal epithelial cells. Hence, serpinE2 may be a potential therapeutic target for colorectal cancer treatment.