Jacques G. Lussier

Gene Expression Profiling of Differentially Expressed Genes in Granulosa Cells of Bovine Dominant Follicles Using Suppression Subtractive Hybridization

Abstract Development of antral follicles beyond 3 to 4 mm in cattle appears as a wave pattern that occurs two to three times during the estrous cycle. Each wave presents a cyclic recruitment of multiple follicles at the 3- to 4-mm stage, followed by the selection of a single follicle that becomes the dominant follicle (DF). The molecular determinants involved in the follicular dominance process remain poorly understood. The objective of the current study was to compare gene expression in granulosa cells (GCs) between growing dominant follicles from Day 5 of the estrous cycle and nonselected small follicles (≤4 mm) using the suppression subtractive hybridization (SSH) approach to identify candidate genes differentially expressed in GCs of the DF. Small follicle cDNAs were subtracted from DF cDNAs (DF-SF) and used to establish a DF GC-subtracted cDNA library. A total of 42 nonredundant cDNAs were identified. Detection of previously identified genes such as CX43, CYP19, INHBA, and SERPINE2 supported the validity of our experimental model and the use of SSH as the method of analysis. For selected genes such as ApoER2, CPD, CSPG2, 14-3-3 epsilon, NR5A2/SF2, RGN/SMP30, and SERPINE2, gene expression profiles were compared by virtual Northern blot or reverse transcriptase-polymerase chain reaction, and results confirmed an increase or induction of their mRNA in GCs of dominant follicles compared with that of small follicles. We conclude that we have identified novel genes (known and unknown) that are up-regulated in bovine GCs that may affect follicular growth, dominance, or both.

Identification of Downregulated Messenger RNAs in Bovine Granulosa Cells of Dominant Follicles Following Stimulation with Human Chorionic Gonadotropin

Abstract 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 32P-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.

Porcine and bovine steroidogenic acute regulatory protein (StAR) gene expression during gestation.

We have generated complete complementary DNA (cDNA) sequences for the porcine steroidogenic acute regulatory protein (StAR) gene, using a combination of genomic PCR amplification and reverse transcription-PCR amplification of pig ovarian cDNA. Porcine StAR cDNA consists of 855 bp and shares 90.2%, 87.3%, 84.3%, and 83.9% homologies with bovine, human, mouse, and rat StAR cDNA at the nucleotide level, and 89.1%, 88.8%, 86.7%, and 86.3% homologies with bovine, human, mouse, and rat StAR protein at the deduced amino acid level. Northern analysis of porcine StAR showed that it is expressed in adult and fetal steroidogenic tissues, including adult testes and ovaries and adult adrenal glands as well as steroidogenic tissues of pregnancy, including developing fetal testes, corpus luteum, and pregnancy, but not the fetal ovary. Major hybridizing bands of 1.8 and 1.1 kilobases were demonstrated. In contrast to human StAR, porcine StAR was not expressed in adult or fetal kidneys. Expression of porcine StAR by the p...

Porcine SRY Promoter Is a Target for Steroidogenic Factor 1

Abstract To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated a 4.5-kilobase (kb) pig SRY 5′ flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (SF-1), and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at −661 base pairs (bps) 5′ from the translation initiation site. Deletion studies of the SRY 5′ flanking sequences in PGR 9E11 cells demonstrated that −1.4 kb of 5′ flanking sequences retained full transcriptional activity compared with the −4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, because deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the −1.4 kb fragment identified two potential binding sites for SF-1, at −1369 and at −290 from the ATG. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse SF-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at −1369 bp and at −290 bp, and that the site at −1369 bp is quantitatively the most important.

High Expression of Bovine α Glutathione S-Transferase (GSTA1, GSTA2) Subunits Is Mainly Associated with Steroidogenically Active Cells and Regulated by Gonadotropins in Bovine Ovarian Follicles1

We have previously shown that a major group of 28–30 kDa proteins decreases after the LH surge in bovine granulosa cells (GC). In the present study, we have characterized two proteins in this group in search of factors that may intervene in folliculogenesis and oocyte maturation. Polyclonal antibodies raised against 28 kDa or 29 kDa bovine GC proteins were used to screen a complementary DNA (cDNA) expression library. This resulted in the characterization of two isoenzyme subunits for α class glutathione S-transferase, named bGSTA1 and bGSTA2. Both bGSTA1 (25.4 kDa, pI 8.9; 791 bp cDNA; GenBank Accession No. BTU49179) and bGSTA2 (25.6 kDa, pI 7.2; 959 bp cDNA; GenBank Accession No. AF027386) have 222 amino acids. The deduced amino acid sequences were compared and showed 82% (bGSTA1) and 74% (bGSTA2) identity to human GSTA1, whereas bGSTA1 and bGSTA2 are 81% identical to each other. The bGSTA2 represents a novel GSTA subunit because it harbors a specific 16 amino acid sequence not found in any other species...

Role of transforming growth factor-β1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.

Expression of Phospholipase A2 Group IVA (PLA2G4A) Is Upregulated by Human Chorionic Gonadotropin in Bovine Granulosa Cells of Ovulatory Follicles

Abstract Prostaglandins are required for the ovulatory process, and their biosynthesis depends on the initial release of arachidonic acid from membrane phospholipids. We hypothesized that phospholipase A2 group IVA (PLA2G4A) expression is upregulated in granulosa cells (GC) at ovulation. We have characterized bovine PLA2G4A cDNA, and investigated its spatiotemporal regulation at the mRNA and protein levels in hCG-induced ovulatory follicles and in vitro, using forskolin-stimulated GC. Regulation of PLA2G4A mRNA expression was studied in GC obtained from bovine follicles collected at different developmental stages: small follicles (2–4 mm), dominant follicles at Day 5 (D5) of the estrous cycle, ovulatory follicles 24 h following injection of hCG, and corpus luteum at D5. PLA2G4A mRNA increased by 14-fold in GC of hCG-stimulated versus dominant follicles (P < 0.0001). Follicular walls obtained from ovulatory follicles recovered at 0, 6, 12, 18, and 24 h post-hCG injection showed an initial 16-fold increase in PLA2G4A transcript at 12 h that reached a 45-fold increase at 24 h, as compared to 0 h (P < 0.0001). Immunoblots of GC extracts showed an initial induction of the PLA2G4A protein at 18 h post-hCG, reaching a maximum at 24 h. Immunohistochemistry observations showed that PLA2G4A signal was mainly observed in mural GC compared to antral GC in hCG-stimulated follicles. Stimulation of cultured bovine GC with 10 μM of forskolin caused an increase in PLA2G4A mRNA and protein. Ovulation is associated with an LH/hCG-dependent induction of PLA2G4A in GC via the adenylyl cyclase/cAMP pathway.

Pre-Sertoli Specific Gene Expression Profiling Reveals Differential Expression of Ppt1 and Brd3 Genes Within the Mouse Genital Ridge at the Time of Sex Determination

Abstract In mammals, testis determination is initiated when the SRY gene is expressed in pre-Sertoli cells of the undifferentiated genital ridge. SRY directs the differentiation of these cells into Sertoli cells and initiates the testis differentiation pathway via currently ill-defined mechanisms. Because Sertoli cells are the first somatic cells to differentiate within the developing testis, it is likely that the signals for orchestrating testis determination are expressed within pre-Sertoli cells. We have previously generated a transgenic mouse line that expresses green fluorescent protein under the control of the pig SRY promoter, thus marking pre-Sertoli cells via fluorescence. We have now used suppression-subtractive hybridization (SSH) to construct a normalized cDNA library derived from fluorescence-activated cell sorting (FACS) purified pre-Sertoli cells taken from 12.0 to 12.5 days postcoitum (dpc) fetal transgenic mouse testes. A total of 35 candidate cDNAs for known genes were identified. Detection of Sf1, a gene known for its role in sex determination as well as Vanin-1, Vcp1, Sparc, and Aldh3a1, four genes previously identified in differential screens as gene overexpressed in developing testis compared with ovary, support the biological validity of our experimental model. Whole-mount in situ hybridization was performed on the 35 candidate genes for qualitative differential expression between male and female genital ridges; six were upregulated in the testis and one was upregulated in the ovary. The expression pattern of two genes, Ppt1 and Brd3, were examined in further detail. We conclude that combining transgenically marked fluorescent cell populations with differential expression screening is useful for cell expression profiling in developmental systems such as sex determination and differentiation.

The serine protease inhibitor serpinE2 is a novel target of ERK signaling involved in human colorectal tumorigenesis

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.

Bovine Activin Receptor Type IIB Messenger Ribonucleic Acid Displays Alternative Splicing Involving a Sequence Homologous to Src-Homology 3 domain Binding Sites1

Activins are implicated in a variety of biological effects, particularly in reproductive processes such as embryonic development and folliculogenesis. Breakthroughs in the elucidation of the activin signal transduction mechanism were achieved with the characterization of the activin receptors, and the recent identification of cytoplasmic factors apparently involved in the signaling process. The present studies were undertaken to further analyze the activin signaling pathway. The complementary DNA coding for the bovine activin receptor type IIB (bActRIIB) was amplified by RT-PCR from corpus luteum and pituitary RNA, and cloned to characterize its role in activin signal transduction. Two complementary DNA isoforms (bAct-RIIB2 and bActRIIB5) were detected, coding for 512 amino acids and 498 amino acids, respectively. The shortest isoform lacked a sequence encoding a 14-amino acid stretch very rich in proline residues, located between the transmembrane region and the intracellular kinase domain. Intron sequen...