Jean Sirois

Ovarian Cell Differentiation: A Cascade of Multiple Hormones, Cellular Signals, and Regulated Genes

During the development of preovulatory follicles, tonic levels of FSH (and steroid) induce expression of aromatase, the LH receptor, and RII beta in a coordinate manner. Despite the similar temporal increase in steady-state levels of mRNA encoding these proteins, the cis-acting DNA elements and trans-acting factors regulating each gene are distinct (Richards, 1993). Whereas the aromatase gene has a TATA motif and a single transcriptional initiation site (Fitzpatrick and Richards, 1993), both the LH receptor (Wang et al., 1992; Tsai-Morris et al., 1993) and RII beta (Kurten et al., 1992; Luo et al., 1992) genes have promoters that are GC rich, lack TATA motifs, and initiate transcription at multiple sites. The aromatase promoter appears to be regulated, in part, by SF-1, a CRE-like region, and possibly another or overlapping region binding an Ad3BP-like factor. The RII beta promoter has a region that binds several nuclear proteins, whose identity is not yet known. Likewise, the LH receptor promoter elements have yet to be clearly defined (Figures 2, 4, and 25; Kurten et al., 1992). FSH can also induce the expression of at least three immediate-early genes that encode novel kinases or kinase-like proteins (Figure 25). One of these is called serum-inducible kinase (snk) (Simmons et al., 1992), another is serum and glucocorticoid regulated kinase (sgk) (Webster et al., 1993), and a third is called pole kinase (Clay et al., 1993). Steady-state levels of snk and sgk mRNA are induced rapidly (within a few hours) by FSH in granulosa cells prior to the appearance of transcripts for aromatase, LH receptor, and RII beta (T. Alliston and J. S. Richards, in preparation). The functional role of these kinases in the initial response of granulosa cells to tonic (not surge) levels of FSH remains to be elucidated. The cellular signaling pathways mediating the effects of the LH surge appear equally or more complex (Fig. 25). Based on data presented herein, as well as on analyses of the cloned and expressed LH receptor (Guderman et al., 1992), it is clear that low concentrations of LH stimulate adenylyl cyclase, cAMP production, and activation of protein kinase A. Higher (surge) concentrations of LH also increase IP3 and activation of protein kinase C. GnRH has been used in several studies to examine the ability of the protein kinase C pathway to mimic effects of high LH.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Cyclooxygenases 1 and 2 and Prostaglandin E Synthase in Bovine Endometrial Tissue During the Estrous Cycle

Abstract In ruminants, endometrial prostaglandin F2α (PGF2α) is responsible for luteolysis and prostaglandin E2 (PGE2) is thought to be involved in maternal recognition of pregnancy. In the present study, healthy uteri were collected from cows at the abattoir, and days of the estrous cycle were determined macroscopically. The uteri were classified into seven groups as Days 1–3, 4–6, 7–9, 10–12, 13–15, 16–18, and 19–21 of the estrous cycle. Endometrial scrapings were collected. The expression of cyclooxygenase (COX)-1 and COX-2 mRNAs and proteins and PGE synthase (PGES) mRNA was analyzed by Northern and Western blot. There was no expression of COX-1, either mRNA or protein, on any day of the estrous cycle. In contrast, COX-2 mRNA and protein were expressed at low and high levels on Days 1–12 and 13–21 of the estrous cycle, respectively. The level of expression of PGES was moderate, low, and high on Days 1–3, 4–12, and 13–21 of the estrous cycle, respectively. There were significant correlations between COX-2 mRNA and protein levels and between COX-2 and PGES mRNA levels. COX-1 mRNA and protein are not expressed on any day of the estrous cycle, whereas COX-2 mRNA and protein and PGES mRNA are differentially expressed and regulated in bovine endometrium during the estrous cycle. COX-2, rather than COX-1, is the primary isoenzyme involved in the endometrial production of prostaglandins, and the COX-2 and PGES pathway is responsible for the endometrial production of PGE2 in the bovine endometrium during the estrous cycle.

THE LATE INDUCTION OF PROSTAGLANDIN G/H SYNTHASE-2 IN EQUINE PREOVULATORY FOLLICLES SUPPORTS ITS ROLE AS A DETERMINANT OF THE OVULATORY PROCESS

PGs are important mediators of the ovulatory process and prostaglandin G/H synthase-2 (PGHS-2) is a key rate-limiting enzyme in the PG biosynthetic pathway. To determine whether PGHS-2 is regulated in equine follicles before ovulation and, if so, to characterize its time course of induction, preovulatory follicles were isolated during estrus, 0, 12, 24, 30, 33, 36, and 39 h after an ovulatory dose of hCG (n = 5 follicles/time point). Cellular extracts were obtained from preparations of follicle wall (theca interna with attached granulosa cells), isolated granulosa cells, and theca interna and were analyzed by Western blot using specific anti-PGHS antibodies. Immunohistochemistry was used to characterize the in situ localization of PGHS-2 protein in preovulatory follicles, and follicular fluid concentrations of PGE2 and PGF were determined. The results showed the induction of PGHS-2, but not PGHS-1, in equine follicles before ovulation. The PGHS-2 protein (72,000 mol wt) was undetectable 0, 12, and 24 h po...

Misregulated Wnt/β-Catenin Signaling Leads to Ovarian Granulosa Cell Tumor Development

Misregulation of the Wnt/beta-catenin signaling pathway is a hallmark of several forms of cancer. Components of the Wnt/beta-catenin pathway are expressed in ovarian granulosa cells; nevertheless, its potential involvement in granulosa cell tumorigenesis has not been examined. To this end, human (n = 6) and equine (n = 18) granulosa cell tumors (GCT) were analyzed for beta-catenin expression by immunohistochemistry. Unlike granulosa cells of normal ovaries, most (15 of 24) GCT samples showed nuclear localization of beta-catenin, suggesting that activation of the Wnt/beta-catenin pathway plays a role in the etiology of GCT. To confirm this hypothesis, Catnb(flox(ex3)/+); Amhr2(cre/+) mice that express a dominant stable beta-catenin mutant in their granulosa cells were generated. These mice developed follicle-like structures containing disorganized, pleiomorphic granulosa by 6 weeks of age. Even in older mice, these follicle-like lesions grew no larger than the size of antral follicles and contained very few proliferating cells. Similar to corpora lutea, the lesions were highly vascularized, although they did not express the luteinization marker Cyp11a1. Catnb(flox(ex3)/+); Amhr2(cre/+) females were also found to be severely subfertile, and fewer corpora lutea were found to form in response to exogenous gonadotropin compared with control mice. In older mice, the ovarian lesions often evolved into GCT, indicating that they represent a pretumoral intermediate stage. The GCT in Catnb(flox(ex3)/+); Amhr2(cre/+) mice featured many histopathologic similarities to the human disease, and prevalence of tumor development attained 57% at 7.5 months of age. Together, these studies show a causal link between misregulated Wnt/beta-catenin signaling and GCT development and provide a novel model system for the study of GCT biology.

Regulation of Cyclooxygenase-2 and Prostaglandin F Synthase Gene Expression by Steroid Hormones and Interferon-τ in Bovine Endometrial Cells1

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-τ (IFN-τ) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-τ (rbIFN-τ) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2α and PG E2 (PGE2) whereas progesterone increased PGF2α secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-τ attenuated both PGF2α and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2α, in stromal cells. Northern blot analysis showed that E2 and rbIFN-τ decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-τ increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-τ decreased PGFS mRNA in both cell types and this was associated wit...

Expression of Cyclo-oxygenase-2 in Naturally Occurring Squamous Cell Carcinomas in Dogs

Squamous cell carcinoma is one of the most common cancers in humans and is also a frequently diagnosed neoplasm in dogs. Induction of cyclo-oxygenase-2 (COX-2), a key rate-limiting enzyme in prostaglandin biosynthesis, has been implicated in the oncogenesis of various cancers in humans, including squamous cell carcinomas. However, expression of COX-2 has not been reported in spontaneous squamous cell carcinomas of non-human species. Canine squamous cell carcinomas share several similarities with the human disease. Therefore, the objective of this study was to determine whether COX isoenzymes were expressed in naturally occurring cases of squamous cell carcinomas in dogs. Canine normal skin (n=4) and squamous cell carcinomas (n=40) were studied by immunohistochemistry and immunoblotting analysis using polyclonal antibodies selective for COX-1 or COX-2. COX-2 was strongly expressed by neoplastic keratinocytes in all cases of squamous cell carcinomas, whereas no COX-2 was detected in normal skin and in the nonneoplastic skin and oral mucosa included in the tumor tissue samples (p < 0.01). Immunoblotting analysis confirmed the restricted expression of COX-2 (72,000–74,000 molecular weight doublet) in squamous cell carcinomas only. In contrast, faint COX-1 staining was found in normal skin and in squamous cell carcinomas. This study demonstrates for the first time that COX-2 is induced in canine squamous cell carcinomas, and provides a new model to investigate the role and regulation of COX-2 gene expression in naturally occurring squamous cell carcinomas.

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.

Expression of Key Prostaglandin Synthases in Equine Endometrium During Late Diestrus and Early Pregnancy

Abstract Luteolysis in domestic species is mediated by the release of luteolytic pulses of prostaglandin (PG) F2α by the uterus at the end of diestrus, which must be suppressed by the conceptus to permit maternal recognition of pregnancy. In many species, including the horse, both the conceptus and the endometrium also synthesize PGE2, which may antagonize PGF2α by playing a luteotropic and/or antiluteolytic role. While the release of PGE2 and PGF2α by the equine endometrium in late diestrus and early pregnancy has been previously studied, the underlying prostaglandin synthase gene regulatory mechanisms remain poorly defined. To resolve this issue, cyclooxygenase-2 (COX-2), microsomal PGE2 synthase (PGES), and PGF2α synthase (PGFS) expression were examined in a series of endometrial biopsies obtained from cycling mares on Days 10, 13, and 15 postovulation, as well as from pregnant mares on Day 15. Quantification of COX-2 expression revealed significant (P < 0.01) increases in both mRNA and protein levels at Day 15 in cycling endometrium relative to other timepoints. Importantly, the level of COX-2 expression in Day 15 pregnant endometrium was found to be comparable with that observed in Day 10 and Day 13 cycling animals, suggesting that the presence of the conceptus blocks the induction of COX-2. Immunohistochemistry demonstrated that the induction of COX-2 expression on Day 15 occurs specifically in surface epithelial cells in cycling animals only. As equine PGFS had not been previously characterized, a 1380-base pair (bp) cDNA transcript was cloned by a combination of reverse transcription-PCR techniques and found to be highly homologous to bovine liver-type PGFS. The pattern of expression observed for the terminal PG synthases was distinct from that of COX-2, as PGES and PGFS mRNA and protein levels were found to be invariant throughout the timecourse and unaffected by pregnancy. Similar to COX-2, however, the PGES and PGFS proteins were found to localize mainly to the surface epithelium. Thus, this study describes for the first time the regulation and spatial distribution of COX-2, PGES, and PGFS expression in equine endometrium in late diestrus, with a marked induction of COX-2 but not of PGES and PGFS expression in uterine epithelial cells at Day 15. Furthermore, the presence of the conceptus was shown to block the induction of COX-2 expression at Day 15, suggesting an important mechanism by which it may suppress uterine PGF2α release and prevent luteolysis during early pregnancy.

Interferon-τ Modulates Phorbol Ester-Induced Production of Prostaglandin and Expression of Cyclooxygenase-2 and Phospholipase-A2 from Bovine Endometrial Cells

Abstract Antiluteolytic actions of bovine interferon-tau (bIFN-τ) require suppression of prostaglandin F2α (PGF2α) production. Our objective was to test whether bIFN-τ could block PGF2α production and synthesis of phospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2) enzymes induced by a protein kinase C (PKC) stimulator (phorbol 12,13 dibutyrate; PDBu). Bovine endometrial epithelial (BEND) cells were treated with PDBu in the presence or absence of bIFN-τ. Medium samples were analyzed for concentrations of PGF2α, whole-cell extracts were analyzed for abundance of PLA2 and COX-2 by immunoblotting, and RNA extracts were examined for steady-state levels of COX-2 mRNA by Northern blotting. The PDBu stimulated production of PGF2α between 3 and 12 h, levels of COX-2 mRNA by 3 h and protein expression of COX-2 and PLA2 by 6 and 12 h, respectively. Added concomitantly with PDBu, bIFN-τ suppressed PGF2α production, steady-state levels of COX-2 mRNA, and expression of COX-2 and PLA2 proteins. Added after a 3-h stimulation with PDBu alone, bIFN-τ suppressed PGF2α production after 1 h. Bovine IFN-τ inhibited intracellular mechanisms responsible for PGF2α production in BEND cells, and this could be through both cytosolic and nuclear actions.

Expression of Prostaglandin G/H Synthase Type 1, but not Type 2, in Human Ovarian Adenocarcinomas

Prostaglandin endoperoxide synthase (PGHS) is a key rate-limiting enzyme in prostaglandin biosynthesis. PGHS has recently been shown to be expressed in human colorectal cancers and in experimental cutaneous papillomas and carcinomas. However, PGHS expression has not been investigated in ovarian cancers. The objectives of this study were to determine whether PGHS isoenzymes are expressed in human ovarian cancer and, if so, to identify which isoform is involved (PGHS-1 and/or PGHS-2) and to characterize its cellular localization. Sixteen human ovarian adenocarcinomas were studied by immunohistochemistry using specific antibodies recognizing PGHS-1 or PGHS-2. Results showed that all adenocarcinomas demonstrated the presence of tumor cells expressing PGHS-1 but not PGHS-2. Patterns of staining of tumor cells varied among different types of adenocarcinomas, with cells presenting either a mostly diffuse cytoplasmic immunoreactivity or, alternatively, a staining mainly concentrated around the nucleus. No correlation between the intensity of the immunostaining and the degree of malignancy of tumors could be established (r −0.03: p>0.05). Immunoblot analysis with PGHS-1-selective antibodies of cell extracts from adenocarcinomas revealed the presence of a characteristic 72,000 Mr immunoreactive band. Therefore, these results show for the first time that PGHS-1 is expressed in human ovarian adenocarcinomas.