Biomedical subjects

J Sirois

Publications and source records attributed to J Sirois.

At least 55 records · Page 3Linked to original sources

PubMed1998

Cloning, developmental expression, and immunohistochemistry of cyclooxygenase 2 in the endometrium during embryo implantation and gestation in the mink (Mustela vison).

Cyclooxygenase (COX) is the first rate-limiting enzyme in the biosynthesis of PGs. There are two isoforms, COX-1, a constitutive enzyme and COX-2, the induced form, products of two different genes. In this study, we report COX-2 complementary DNA cloning, uterine expression, and immunohistochemical localization in the mink uterus during postimplantation gestation. The open reading frame of mink COX-2 contains 1812 nucleotides encoding 604 amino acids. The homologies are 86%, 83%, 83%, 83%, 85%, and 84% in nucleotides and 86%, 87%, 87%, 85%, 86%, and 88% in amino acids with human, mouse, rat, guinea pig, sheep, and rabbit, respectively. All domains associated with biological activity are conserved in the mink. Northern analysis revealed a transcript of 4.2 kb for COX-2 in mink uterus and adrenal. Semiquantitative RT-PCR showed that COX-2 messenger RNA is not present during diapause. The abundance of COX-2 messenger RNA reached its maxima (P < 0.05) on days 3-5 of postimplantation, gradually decreased through day 9, and was not present thereafter. By immunohistochemistry, COX-2 was present in uterine epithelium, stroma, and necks of endometrial glands at sites of implantation. COX-2 expression appears to be induced in the endometrium by the embryo and may play a role in implantation and placentation in the mink.

Amino Acid Sequence↗

PubMed1997

Effects of dexamethasone administration to diestrus cows on systemic progesterone, estrogen and uterine cyclooxygenase production.

Parenteral administration of dexamethasone to diestrus cattle can extend the length of the natural estrous cycle. In mice, dexamethasone has been shown to inhibit production of the second isozyme of the cyclooxygenase (COX) enzyme (a rate limiting enzyme in prostaglandin formation). Therefore, the purpose of this study was to determine the effect of dexamethasone on estrous cycle length and COX-1 and -2 production by the uterine endometrium of cyclic cattle. Nine crossbred beef cows that exhibited two previous normal estrous cycles were randomly assigned to two treatments; a control group administered intramuscular injections of vehicle, and a dexamethasone group administered 8 mg of dexamethasone (Azium, Schering Corp., Kenilworth, NJ). Both groups received twice daily injections on day 13-22 of the treatment cycle. Uterine endometrial biopsies were collected on days 16, 19 and 22 of the treatment cycle. Blood samples were collected daily on day 13-22 of the treatment cycle for plasma progesterone and estradiol concentrations.

Animals↗

PubMed1997

Prostaglandin G/H synthase-2 is expressed in bovine preovulatory follicles after the endogenous surge of luteinizing hormone.

To determine whether prostaglandin G/H synthase-2 (PGHS-2) induction occurs under physiological conditions, heifers were ovariectomized and preovulatory follicles were isolated 0 (n = 4), 18 (n = 4), and 24 h (n = 4) after the onset of estrus. The time of the LH surge was determined in blood samples collected every 4 h pre- and postestrus. Preparations of follicle wall and isolated theca interna and granulosa cells were analyzed for PGHS-2 protein and mRNA by immunoblot and Northern blot, respectively. Immunohistochemistry was used to document the in situ localization of PGHS-2 protein, and follicular fluid concentrations of prostaglandin (PG) E2 and PGF2alpha were determined to monitor changes in PG synthetic activities. Results showed that PGHS-2 mRNA (4.0 kilobases) and protein (74000 Mr) were absent in preovulatory follicles isolated at the onset of estrus, low in follicles obtained 18 h after estrus (16.0 +/- 1.2 h post-LH surge), and markedly induced 24 h postestrus (20 +/- 0 h post-LH surge). Immunoblot and immunohistochemical analyses revealed that PGHS-2 protein was selectively induced in granulosa cells. Follicular fluid concentrations of PGE2 and PGF2alpha increased significantly (p < 0.01) between 0 and 24 h after estrus (from 2.8 +/- 0.2 to 87.9 +/- 30.9 ng/ml for PGE2; from 0.05 +/- 0.02 to 68.9 +/- 23.6 ng/ml for PGF2alpha). Collectively, these results clearly demonstrate that the induction of PGHS-2 in bovine preovulatory follicles is a physiological event that occurs after the endogenous LH surge.

Animals↗

PubMed1997

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 post-hCG, first became apparent at 30 h, and reached maximal levels 39 h after hCG treatment. The induction of follicular PGHS-2 was localized exclusively in granulosa cells, and a pronounced staining was observed in the perinuclear region. Follicular fluid concentrations of PGE2 and PGF were low and not different between 0-33 h, but levels were increased at 36 and 39 h post-hCG (P < 0.01). Thus, the time course of PGHS-2 induction in equine follicles (30 h post-hCG) is clearly distinct from those previously observed in rat (4 h post-hCG) and bovine (18 h post-hCG) preovulatory follicles. Interestingly, in all three species, the interval from PGHS-2 induction to follicular rupture is highly conserved (approximately 10 h). Therefore, the progressively delayed expression of PGHS-2 in species with longer ovulatory processes supports its role as a molecular determinant of the species-specific length of the ovulatory process.

Analysis of Variance↗

PubMed1996

Regulation of P-selectin expression by inflammatory mediators in canine jugular endothelial cells.

Canine endothelial cells express the adhesion molecule P-selectin to mediate the initial attachment of leukocytes to the vessel wall. Although it is known that agents like histamine and thrombin stimulate the surface expression of P-selectin, the effect of inflammatory mediators and cytokines such as lipopolysaccharides (LPS), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) on canine P-selectin expression has not been investigated. Therefore, the objective of this study was to analyze the regulation of P-selectin messenger RNA (mRNA) and protein by these cytokines in canine endothelial cells isolated from jugular veins. Analyses of cytoplasmic RNA by Northern blotting showed that stimulation of culture endothelial cells with either LPS (100 ng/ml) or recombinant human TNF-alpha (30 U/ml) for 3 or 6 hours significantly increased (P < 0.05) steady-state levels of mRNA for P-selectin (3.8- +/- 1.0- and 3.0- +/- 0.4-fold increase for LPS at 3 and 6 hours, respectively, and 2.5- +/- 0.8- and 2.7- +/- 0.9-fold increase for TNF-alpha at 3 and 6 hours, respectively). P-selectin mRNA had decreased by 48 hours to levels found in unstimulated cells. In contrast, human IL-1 beta had no effect on P-selectin mRNA. Increased levels of mRNA with LPS stimulation were associated with the synthesis of new protein, as demonstrated by the positive staining in LPS-stimulated cells using immunocytochemistry with a monoclonal antibody against canine P-selectin (MD3). These results reveal that important inflammatory mediators and cytokines such as LPS and TNF-alpha induce the synthesis of new P-selectin and suggest that this process could represent a means of sustaining local leukocyte recruitment for several hours during an acute inflammatory reaction.

Analysis of Variance↗

PubMed1995

Oxytocin in mares: lack of evidence for oxytocin production by or action on preovulatory follicles.

It is not known whether the equine preovulatory follicle produces oxytocin or is a target tissue for oxytocin, as has been reported for other species, especially ruminants. Bovine granulosa cells secrete oxytocin, and oxytocin modulates the production of progesterone by granulosa cells in vitro. We examined whether oxytocin plays a comparable role in the equine preovulatory follicle. To test the hypothesis that the equine preovulatory follicle produces oxytocin during estrus and that its production increases in late estrus, preovulatory follicles were isolated during early (Days 1 to 2; n = 4) and late (Days 4 to 5; n = 4) estrus. Granulosa cells, pieces of theca interna and pieces of follicle wall (theca with attached granulosa cells) were cultured for 3 d with or without equine gonadotropins. Culture media were collected, replaced at 3, 6, 12, 24, 48, and 72 hr of culture, and assayed for oxytocin. Granulosa cells from preovulatory follicles secreted negligible amounts of oxytocin during 3 d of culture, irrespective of gonadotropin treatment or stage of estrus. Likewise, negligible amounts of oxytocin were measured in theca and follicle wall cultures at both developmental stages, in the presence or absence of gonadotropins. Furthermore, follicular fluid from early or late estrous follicles contained only negligible amounts of oxytocin. To determine if oxytocin affects steroidogenesis by equine granulosa cells, granulosa cells from follicles obtained on Day 2 of estrus were cultured with graded doses of oxytocin (0, 1, 10, 100, and 1,000 ng/ml) in defined medium supplemented with testosterone (0.5 microM) and culture media were assayed for estradiol-17 beta and progesterone. Estradiol was secreted throughout the culture period, and its production was not significantly affected by oxytocin treatment (P > 0.05). Progesterone secretion was relatively low during the first 24 hr of culture, increased dramatically on the second day of culture, and remained high through the third day. No dose of oxytocin had a significant effect on progesterone secretion (P > 0.05). In conclusion, the results indicate that equine preovulatory follicles, isolated during early or late estrus, are neither a source of oxytocin nor a target for oxytocin action on steroidogenesis. Although ovarian oxytocin appears to play a role in regulating follicular function in some other mammalian species, our data provide no support for such a role for oxytocin in mares.

Animals↗

PubMed1995

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)

Animals↗

PubMed1994

Induction of prostaglandin endoperoxide synthase-2 by human chorionic gonadotropin in bovine preovulatory follicles in vivo.

Gonadotropins have recently been shown to induce prostaglandin endoperoxide synthase-2 (PGHS-2) in rat preovulatory follicles before ovulation, but hormonal induction of this enzyme has not yet been reported in preovulatory follicles of any other species. To determine whether the selective induction of PGHS-2 by the ovulatory gonadotropin surge is a molecular process that has been conserved in other mammalian species with different reproductive cycles, the regulation of PGHS isoforms was studied in bovine preovulatory follicles isolated 0, 6, 12, 18, 20, 22, 24, and 26 h after an ovulatory dose of hCG. Each follicle was dissected into three preparations: the intact follicle wall (i.e. theca interna with attached granulosa cells), theca interna, and isolated granulosa cells. Solubilized cell extracts were obtained from each preparation and analyzed by Western blots, using polyclonal antibodies that selectively recognize PGHS-2 and PGHS-1 isoforms. RNA extracts were prepared and analyzed by Northern blots, using a mouse PGHS-2 complementary DNA. The results indicated that the expression of PGHS-2 messenger RNA (mRNA) and protein, but not that of PGHS-1, was regulated by hCG in bovine preovulatory follicles before ovulation. Levels of PGHS-2 protein (mol wt, 74,000) in the follicle wall were undetectable at 0, 6, and 12 h, but increased dramatically after 18 h to reach maximal levels 24 h post-hCG treatment. Northern blots revealed a similar temporal induction of PGHS-2 mRNA (4.0 kilobases). Analyses of isolated preparations of theca interna and granulosa cells clearly showed that PGHS-2 was expressed exclusively in granulosa cells and not in theca interna. Significant increases in follicular fluid concentrations of prostaglandins E2 and F were associated with the induction of follicular PGHS-2 protein and mRNA. Thus, this study establishes that the induction by gonadotropin of PGHS-2 in granulosa cells before ovulation appears to be a conserved mechanism by which the synthesis of prostaglandins necessary for the ovulation process is regulated in different species. However, the striking difference between the time course of PGHS-2 induction previously reported in rat follicles (4 h post-hCG treatment) and that observed in bovine follicles (18 h post-hCG treatment) suggests that the control of PGHS-2 expression could be one of the determinants involved in dictating the species-specific progression (length) of the ovulation process.

Animals↗

PubMed1993

Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element.

The promoter of the rat prostaglandin endoperoxide synthase 2 (PGS-2) gene has recently been shown to confer gonadotropic hormone (follicle-stimulating hormone (FSH), luteinizing hormone (LH), cAMP) inducibility when ligated to a CAT (chloramphenicol acetyltransferase) reporter gene and transfected into primary cultures of differentiated granulosa cells. To delineate cis-acting elements and trans-activating factors mediating this response, deletions of the active promoter region (-192/-53 base pairs upstream of the transcriptional start site) were tested for their ability to bind protein of granulosa cell nuclear extracts and activate reporter gene activity. Electrophoretic mobility shift assays revealed that the DNA subregion -142/-120 inhibited protein/DNA binding observed between granulosa cell nuclear extracts and the labeled PGS-2 fragment -192/-53. The subregion -142/-120 acting element C/EBP beta, 5'-TTATGCAAT-3'. Point mutations within the C/EBP beta element abolished protein/DNA binding and resulted in a 50% loss of forskolin/LH/FSH inducibility of reporter gene activity. C/EBP beta mRNA and protein were induced rapidly in granulosa cells in vivo by an ovulatory dose of human chorionic gonadotropin (hCG). Collectively, these results indicate that C/EPB beta appears to play a key role in regulating induction of the PGS-2 gene in granulosa cells prior to ovulation.

Animals↗

PubMed1993

Characterization and hormonal regulation of the promoter of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Identification of functional and protein-binding regions.

Prostaglandin endoperoxide synthase isoform 2 (PGS-2) mRNA and protein are transiently induced by gonadotropins in granulosa cells of preovulatory follicles prior to ovulation. To better understand the hormonal regulation of the rat PGS-2 (rPGS-2) gene in these cells, genomic clones containing rPGS-2 as well as up to 6 kilobases of 5'-flanking DNA were isolated by screening a rat liver genomic library with a labeled 5'-fragment of the mouse PGS-2 cDNA. Primer extension analysis using ovarian follicular mRNA identified the presence of a single rPGS-2 transcription initiation site located 144 nucleotides upstream of the ATG translation initiation codon. To test for promoter activity within the 5'-flanking region of the rPGS-2 gene, a genomic fragment, -2698/32 (1 = cap site), as well as a series of 5'-deletion mutants, were fused upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into primary cultures of granulosa cells. Forskolin (7.5 microM), follicle-stimulating hormone (500 ng/ml) and luteinizing hormone (500 ng/ml) induced CAT activity following transfection with the -2698/32PGS.CAT, whereas gonadotropin-releasing hormone (10(-6) M) and interleukin-1 beta (30 ng/ml) had no effect. Deletion mutants delineated the region spanning from -192 to -54 of the transcription start site to be essential for both basal and forskolin-regulated expression of the reporter gene. The same DNA fragment (-192/-54) exhibited specific binding to granulosa cell nuclear extract proteins as analyzed by electrophoretic mobility shift assays. Additional specific bands were observed in extracts prepared from granulosa cells exposed to an ovulatory dose of gonadotropin. Collectively, these results provide the first structural and functional evidence that the transcriptional regulation of the rat PGS-2 gene by gonadotropins and forskolin in granulosa cells involves 5'-flanking DNA sequences, specifically a region between -192 and -54 of the transcription initiation site.

Animals↗

PubMed1993

Induction of prostaglandin endoperoxide synthase isoform-2 in ovine cotyledonary tissues during late gestation.

The present study was designed to characterize the developmental pattern of specific prostaglandin endoperoxide synthase (PGS) isoform immunoreactivity and activity in tissues of fetal origin during the last half of gestation in sheep. Fetal amnion, allantochorion, and cotyledons were collected under halothane general anesthesia on days 79-80, 105-108, 120-122, 128-131, and 140-145 (term) of pregnancy. Solubilized extracts were prepared and analyzed by immunoblots using anti-PGS antibodies previously shown to recognize PGS isoform-1 and -2 (PGS-1 and PGS-2). PGS activity from cotyledon microsomes was assayed by the measurement of prostaglandin E2 (PGE2) production under initial velocity conditions. All fetal tissues contained PGS-1 at each of the stages of gestation examined, with minimal regulation of this isoform from 79-144 days gestation. In contrast, PGS-2 increased gradually in the cotyledons from 120-139 days gestation, with the most marked expression observed at term. PGS-2 was not detected in amnion or allantochorion. PGS activity in cotyledons increased (P < 0.01) in parallel with immunoreactive PGS-2 levels; indicating that PGS-2, rather than PGS-1, is associated with increased PG synthesis in this tissue. Both the activity (n = 5/group) and the amount of PGS-2 increased significantly from 105-108 days gestation to term (P < 0.01). We conclude that the increase in PGS that occurs at term in sheep is predominantly active PGS-2 localized to fetal cotyledons.

Allantois↗

PubMed1993

Effect of oestradiol on oxytocin-stimulated prostaglandin F2 alpha release in mares.

The objective of this study was to clarify the role of oestradiol in luteal function by examining its effect on the oxytocin stimulation of 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM) concentrations in cyclic mares. In the first experiment, three groups of mares (4 per group) were given a bolus injection of 17 alpha-oestradiol (1 mg), oestradiol (1 mg) or vehicle on days 7, 9, 11, 13 and 15 of the cycle. Six hours later the mares were challenged with 10 iu oxytocin intravenously and frequent blood samples were taken from 15 min before to 15 min after for measurement of PGFM. Results showed a significant stimulatory effect of oestradiol (five times greater than controls at day 11; P < 0.05), but not of 17 alpha-oestradiol, on the oxytocin stimulation of PGFM. As a relatively large dose was given systemically in this experiment, a second experiment was performed to introduce a dose that was more physiological into the uterus. Small Silastic spheres (1 cm diameter) were impregnated with or without oestradiol at a concentration that gave a release rate similar to that of embryos at day 12 (10 ng h-1). These were inserted (one per mare) into the uterus of two groups of mares (five per group) on day 7. The mares were challenged with oxytocin on days 9, 11, 13 and 15 of the cycle and blood samples were taken as before for determination of PGFM. The results showed that oestradiol enhanced (four times greater than controls at day 13; P < 0.05) the oxytocin stimulation of PGFM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles. Induction in vivo and in vitro.

Recent purification and amino-terminal analysis of the isoform of prostaglandin endoperoxide H synthase (PGS) induced in rat preovulatory follicles by gonadotropins identified it as a novel distinct isoform of PGS (rPGSi) which exhibited a high degree of homology to the deduced sequence of PGS-related cDNAs recently cloned in chicken and mice. To further verify the similarities of these novel gene products and to examine the hormonal regulation of rPGSi mRNA in ovarian cells, three different in vivo and in vitro models were used. Northern blots using a cDNA encoding the mouse homologue of rPGSi detected a 4.4-kilobase transcript which was rapidly but transiently induced in granulosa cells of preovulatory follicles exposed in vivo to an ovulatory dose of human chorionic gonadotropin. The rPGSi mRNA was undetectable at 0 h, peaked 4 h after human chorionic gonadotropin, and had almost disappeared by 6 h. Increases in rPGSi protein (immunoblots) lagged by about 1 h, peaked at 5 h, and remained present at 11 h. PGSi mRNA and protein were also induced in a time- and dose-dependent manner when preovulatory follicles were isolated and incubated with elevated levels of follicle-stimulating hormone (500 ng/ml) or luteinizing hormone (500 ng/ml), or when differentiated granulosa cell cultures were stimulated with follicle-stimulating hormone, luteinizing hormone, or with gonadotropin-releasing hormone (10(-6) M). In both in vitro systems, rPGSi mRNA peaked at 4-5 h. When the same RNA samples were probed with the mouse cDNA encoding the other PGS isoform, no mRNA transcripts (2.8 kilobases) were observed. These results show for the first time that a rapid and transient induction of mRNA encoding a novel PGS enzyme occurs in granulosa cells of preovulatory follicles prior to ovulation and that results in vitro closely mimicked those in vivo and thereby provide models for studying the molecular mechanisms of rPGSi gene expression.

Animals↗

PubMed1992

Purification and characterization of a novel, distinct isoform of prostaglandin endoperoxide synthase induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles.

To purify and characterize the isoform of prostaglandin endoperoxide synthase (rPGSi) induced by human chorionic gonadotropin in granulosa cells of rat preovulatory follicles, solubilized cell extracts were subjected to anionic exchange chromatography, column fractions were resolved by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and rPGSi was visualized by immunoblotting and silver staining techniques. Immunoreactive rPGSi and peroxidase activity co-eluted at pH 6.5 and 6.0. Amino-terminal amino acid sequence of three immunoreactive rPGSi bands (Mr = 72,000, 70,000, and 59,000) were identical, Mr = 59,000 being a proteolytic fragment. Alignment of the amino-terminal sequences of rPGSi with those of ovine PGS (oPGS) indicated that only 15 of 26 residues were identical (58% identity). In contrast, rPGSi was closely related to the deduced amino acid sequence of PGS-related cDNA clones isolated from chicken and mouse cell lines, with 77% (20/26 residues) and 96% (25/26 residues) identity, respectively. Whereas tryptic digests of oPGS generated fragments of Mr = 38,000 and Mr = 33,000, only a small peptide appeared cleaved from the carboxyl terminus of rPGSi. Peroxidase activity of partially purified rPGSi exhibited lower apparent Km and maximal velocity than that of oPGS. Collectively, these results document the existence of a novel rat PGS isoform (based on purification, enzymatic activity, and amino-terminal amino acid sequence) which is hormonally induced and obligatory for a known biological process, ovulation.

Amino Acid Sequence↗

PubMed1991

Steroidogenesis by equine preovulatory follicles: relative roles of theca interna and granulosa cells.

Estrous cycles in mares have several unique characteristics, including the presence of a long period of estrus and the absence of a typical LH surge. Like follicles of other species, equine preovulatory follicles are characterized by their ability to secrete large amounts of 17 beta-estradiol, but it is not clear which follicular cell type is responsible for estradiol synthesis in mares. To better understand the relative roles of theca interna and granulosa cells in follicular steroidogenesis, presumptive ovulatory follicles were obtained from mares during early estrus (first or second day of estrus; n = 4) and during late estrus (fourth or fifth day of estrus; n = 4). Preparations of theca interna and granulosa cells were cultured for 3 days in medium with or without equine LH, FSH, LH plus FSH, or CG (100 ng/ml) in the presence or absence of 0.5 microM testosterone, and culture media were assayed for progesterone, androstenedione, and 17 beta-estradiol. Progesterone was the predominant steroid secreted by granulosa cells in the absence of exogenous testosterone. Its accumulation was significantly higher in cultures of granulosa cells from late vs. early estrus (P less than 0.05), and all gonadotropins stimulated progesterone secretion at both stages of follicular development (P less than 0.05). In contrast, granulosa cells secreted very low amounts of androstenedione in vitro, and only very small amounts of 17 beta-estradiol were produced when cells were cultured in medium without testosterone. However, the addition of testosterone caused a 170-fold increase over control values in estradiol accumulation over 3 days of culture (P less than 0.0001), clearly indicating the presence of a very active aromatase enzyme system in equine granulosa cells. Steroid secretion by theca interna differed in several respects from secretion by granulosa cells. Theca interna from early and late estrous follicles secreted negligible amounts of progesterone in vitro, and equine gonadotropins had no effect on its secretion. Also, theca interna secreted only small amounts of estradiol in vitro, and its accumulation was not increased by the addition of exogenous testosterone. Also, in contrast to granulosa cell cultures, androstenedione was the predominant steroid secreted by theca interna from early and late estrous follicles. In conclusion, this study does not support the current model of equine follicular steroidogenesis, which holds that 17 beta-estradiol biosynthesis derives primarily from the theca interna layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Follicle selection in domestic ruminants.

Ultrasound imaging has shown that cattle exhibit 2 or 3 waves of follicular development during an oestrous cycle. The waves consist of the contemporaneous appearance, about every 7 days, of a group of follicles > or = 5 mm in diameter. One follicle gradually becomes larger than the rest (i.e. dominant). There are several lines of evidence suggesting that the waves occur regularly under conditions of basal LH and FSH. (1) Cycles with 3 waves of follicular development are longer and have longer luteal phases than do cycles with 2 waves, indicating that the number of waves in a cycle is determined by the time of luteal regression. (2) Cycles with 4 or 5 waves of follicular development can occur when the luteal phase is artificially prolonged with exogenous progesterone. (3) Waves of follicular development occur during pregnancy. However, the secondary surge of FSH may be important in initiating new follicular recruitment after ovulation, since suppression of the secondary surge delays the first wave of follicular development. Follicles are functionally dominant (capable of ovulating after luteal regression) while they are still growing and early during their plateau in growth. Functional dominance is lost some time between the early and late plateau phases, while the follicle is still morphologically dominant (i.e. the largest follicle). The factors that lead to dominance of one follicle and the mechanisms that suppress the growth of subordinate follicles are not well understood. When the luteal phase is artificially extended with low doses of exogenous progesterone, the normal pattern of follicular development is altered and the ovulatory follicle grows for a prolonged period of time. This finding indicates that subtle changes in the hormonal milieu can dramatically alter follicular dynamics and that the experimental model of prolonged dominance may be useful in studying the mechanisms of follicular dominance. In contrast, patterns of follicular development in sheep must be assessed in more indirect ways, but sheep offer the advantage of breeds that differ in ovulation rate. Correlation of the endocrine environment with ovulation rate in this species provides a valuable approach to understanding the mechanisms controlling follicle selection and ovulation rate. It has been suggested that in some species a high ovulation rate is achieved by increased recruitment, whereas in others there is increased selection. There is evidence that oestradiol is involved in regulating the number of dominant follicles in sheep. Follicular recruitment requires the presence of gonadotrophins, particularly FSH. In general, the mechanisms that regulate follicular selection and dominance in domestic ruminants are not well understood. Further experiments may determine the relative roles of paracrine factors and ovarian-pituitary-hypothalamic interactions in regulation of follicular selection and dominance in cattle and sheep.

Animals↗

PubMed1990

Lengthening the bovine estrous cycle with low levels of exogenous progesterone: a model for studying ovarian follicular dominance.

In cattle the development of ovarian follicles greater than or equal to 5 mm occurs in waves, with either two or three waves per estrous cycle. To increase our understanding of the control of follicular dynamics in cattle, the present study was designed to characterize the pattern of follicular development during artificially lengthened estrous cycles. Cycles were lengthened by intravaginal insertion of Silastic devices containing progesterone [Controlled Internal Drug Release devices (CIDRs)]. Control heifers (group 1) received blank devices, whereas treated heifers received one (group 2) or two CIDRs (group 3) from days 14 to 28 after estrus. In groups 2 and 3, the insertion of CIDRs prevented return to estrus at the normal time and increased cycle length as compared to the control group (30.0 +/- 0.0 and 31.0 +/- 0.3 vs. 21.0 +/- 0.7 days, respectively, P less than 0.05). After natural luteolysis and between days 22 and 28 of cycle, progesterone concentrations were maintained at lower levels in group 2 (range = 0.9-2.1 ng/ml) than in group 3 (range = 3.7-4.9 ng/ml, P less than 0.003). Follicular development and regression were monitored daily by ultrasonography. The number of follicular waves per cycle was identical in groups 1 and 2 (2.7 waves per cycle), despite the significantly longer cycles in group 2. In group 2, the presence of one CIDR altered the normal pattern of follicular development by promoting the prolonged growth of the ovulatory follicle, and associated with it, a complete absence of follicular recruitment. When compared to ovulatory follicles in controls (group 1), ovulatory follicles in group 2 were detected on the ovaries for a longer time (1.8-fold), reached a greater maximal size (1.4-fold), and were dominant for a longer time (3-fold). Heifers in group 3 had significantly more follicular waves per cycle than groups 1 and 2 (3.8 vs. 2.7 waves per cycle, respectively, P less than 0.05), due to the production of additional follicular waves during the lengthened cycle in three of six heifers. The other three heifers in group 3 showed patterns of follicular development similar to those of group 2. All heifers in the control group had normal preovulatory rises in estradiol and LH. During the period of treatment (days 14-28), 17 beta-estradiol concentrations were higher in heifers in group 2 (lower progesterone levels) than in heifers in group 3 (higher progesterone levels; P less than 0.0001). No differences were observed in basal LH concentrations between groups 2 and 3.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Developmental changes in steroidogenesis by equine preovulatory follicles: effects of equine LH, FSH, and CG.

Ovulation in mares is preceded by a long and variable estrous period. The differentiation of equine preovulatory follicles with respect to steroidogenic capacity and responsiveness to equine gonadotropins was studied by culturing pieces of follicle wall (FW = theca + attached granulosa cells) from preovulatory follicles isolated during late diestrus (day 14 of cycle, n = 5 mares), early estrus (1st-2nd day of estrus, n = 6) or late estrus (4th or 5th day of estrus, n = 6). FW was cultured with or without equine LH, FSH, LH + FSH, or CG (10 or 100 ng/ml) and medium was collected and replaced at 3, 6, 12, 24, 48, and 72 h of culture. Follicular fluid from presumptive ovulatory follicles and medium from FW cultures were assayed for progesterone, androstenedione, and estradiol-17 beta. The cumulative secretion of all three steroids after 72 h of culture was significantly lower in FW isolated during late diestrus (P less than 0.05) as compared with early or late estrus. Maximal progesterone secretion was observed with FW from late estrus whereas maximal androstenedione and estradiol secretion in vitro occurred with FW from early estrus. In contrast to results obtained in vitro, concentrations of progesterone in follicular fluid were not different among stages of follicular development, and concentrations of androstenedione and estradiol in follicular fluid were maximal in late estrous follicles. Equine gonadotropins had their greatest stimulatory effect on steroidogenesis with FW obtained during late diestrus. As compared with controls, the addition of LH, FSH, or LH + FSH (100 ng/ml) increased progesterone secretion by FW from late diestrus (48x, 64x, and 58x, respectively, P less than 0.01), early estrus (24x, 32x, and 36x, P less than 0.01) and late estrus (9x, 9x, and 9x, P less than 0.01). Equine LH and FSH also increased androstenedione secretion by follicles obtained during diestrus and estrus. In contrast, estradiol secretion showed a more rapid loss in responsiveness to gonadotropin stimulation, with both LH and FSH stimulating estradiol secretion by FW from late diestrous follicles (P less than 0.01), but neither stimulating FW from early or late estrous follicles. Overall, eCG was a less potent stimulator of steroidogenesis in vitro than LH and FSH.(ABSTRACT TRUNCATED AT 400 WORDS)

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