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1.
J Neuroendocrinol ; 24(5): 737-47, 2012 May.
Article in English | MEDLINE | ID: mdl-22172059

ABSTRACT

Gonadotrophin-releasing hormone (GnRH) agonists are used to treat gonadal steroid-dependent disorders in humans and to contracept animals. These agonists are considered to work by desensitising gonadotrophs to GnRH, thereby suppressing follicle-stimulating hormone (FSH) and luteinising hormone (LH) secretion. It is not known whether changes occur in the cellular composition of the pituitary gland after chronic GnRH agonist exposure. Adult male Sprague-Dawley rats were treated with a sham, deslorelin, or deslorelin plus testosterone implant for 41.0 ± 0.6 days. In a second experiment, rats were castrated and treated with deslorelin and/or testosterone. Pituitary sections were labelled immunocytochemically for FSHß and LHß, or gonadotrophin α subunit (αGSU). Deslorelin suppressed testis weight by two-thirds and reduced plasma FSH and LH in intact rats. Deslorelin decreased the percentage of gonadotrophs, although the effect was specific to the FSHß-immunoreactive (-ir) cells. Testosterone did not reverse the deslorelin-induced reduction in the overall gonadotroph population. However, in the presence of testosterone, the proportion of gonadotrophs that was FSHß-ir increased in the remaining gonadotrophs. There was no effect of treatment on the total LHß-ir cell population, although the loss of FSHß in bi-hormonal cells increased the proportion of mono-hormonal LHß-ir gonadotrophs. The castration-induced plasma LH and FSH increases were suppressed by deslorelin, testosterone or both. Castration increased both LH-ir and FSH-ir without increasing the overall gonadotroph population, thus increasing the proportion of bi-hormonal cells. Deslorelin suppressed these increases. Testosterone increased FSH-ir in deslorelin-treated castrate rats. Deslorelin did not affect αGSU immunoreactivity, suggesting that the gonadotroph population per se is not eliminated by deslorelin, although the ability of gonadotrophs to synthesise FSHß is compromised. We hypothesise that the FSH dominant suppression may be central to the long-term contraceptive efficacy of deslorelin in the male.


Subject(s)
Follicle Stimulating Hormone/metabolism , Triptorelin Pamoate/analogs & derivatives , Animals , Down-Regulation/drug effects , Follicle Stimulating Hormone/blood , Gonadotrophs/drug effects , Gonadotrophs/metabolism , Gonadotropin-Releasing Hormone/agonists , Hormone Antagonists/pharmacology , Immunohistochemistry , Male , Orchiectomy , Organ Size/drug effects , Pituitary Gland/anatomy & histology , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Rats , Rats, Sprague-Dawley , Testis/anatomy & histology , Testis/drug effects , Testis/metabolism , Time Factors , Triptorelin Pamoate/pharmacology
2.
Soc Reprod Fertil Suppl ; 67: 189-201, 2010.
Article in English | MEDLINE | ID: mdl-21755673

ABSTRACT

The ovulatory process is extraordinary in that it constitutes a hormone-induced injury. Gonadotropin delivered via the follicular vascular wreath stimulates secretion of plasminogen activator by contiguous ovarian surface epithelial cells. A consequent elevation in interstitial plasmin activates collagenases and cleaves tumor necrosis factor alpha from its anchors on endothelium. Collagen fibril degradation and cellular death at the apex of the preovulatory follicle are hallmarks of impending ovulation. Follicular contractions rupture the weakened fabric at the apex, and the ovum, which has been disconnected from the underlying granulosa, is expelled; these components of the cascade are prostaglandin-mediated. Ovulation is required for fertility; unfortunately, it imparts a cancer risk to the ovarian surface epithelium. DNA-damaging reactive oxygen species are generated by inflammatory cells attracted into the vicinity of the ovulatory stigma. An ischemia-reperfusion flux coincident with ovulation and wound repair also contributes to genotoxicity. Potentially mutagenic lesions in DNA are normally reconciled by TP53 tumor suppressor-dependent cell-cycle arrest and base excision repair mechanisms; it is a unifocal escape that could be problematic. Epithelial ovarian cancer is a deadly insidious disease because it typically remains asymptomatic until it has metastasized to vital abdominal organs.


Subject(s)
Ovary/physiology , Ovulation/physiology , Sheep/physiology , Animals , Antioxidants , Female , Gene Expression Regulation, Neoplastic , Male , Models, Biological , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Ovary/metabolism , Ovary/pathology , Progesterone/metabolism , Prostaglandins/physiology
3.
J Anim Sci ; 82(9): 2548-57, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15446470

ABSTRACT

The goal of this study was to determine the effects of short-term feed withdrawal on reproductive and metabolic hormones during the luteal phase of the estrous cycle in mature ewes. Mature ewes observed in estrus were assigned randomly to control and fasted groups (n = 10 per group Trials 1 and 2). For Trials 1 and 2, control ewes had ad libitum access to feed, whereas fasted ewes were not fed from d 7 through 11 of their estrous cycle; on d 12, all ewes were treated with 10 mg of PGF2alpha, and fasted ewes were gvien ad libitum access to feed. For Trial 1, blood samples were collected daily through fasting and at 2-h intervals following PGF2alpha for 72 h. Serum concentrations of insulin (P < or = 0.002) and IGF-I (P < or = 0.01), but not GH (P > or = 0.60), were decreased during fasting compared with fed ewes. Serum concentrations of 29 (P = 0.02) and 34 kDa (P = 0.04) IGFBP were greater in fasted ewes at 96 h after initiation of fasting than in control ewes. Two control and four fasted ewes in Trial 1 did not exhibit a preovulatory surge release of LH by 72 h. Therefore, Trial 2 was conducted so that the timing of the LH surge could be predicted following the collection of blood samples at 2-h intervals for 112 h and then at 6-h intervals until 178 h following PGF2alpha administration and realimentation. The magnitude of the preovulatory LH surge in Trial 2 was decreased (P = 0.009) and delayed (P = 0.04), and serum concentrations of estradiol were diminished (P < or = 0.03) 12 h before the LH surge in fasted ewes. Ovulation rates were not influenced (P > or = 0.32) by fasting in Trials 1 and 2. Serum concentrations of progesterone in both Trials 1 and 2 were, however, greater (P < 0.001) in fasted than in control ewes. A third trial with ovariectomized ewes was conducted to determine whether the increased serum concentrations of progesterone observed in fasted ewes during Trials 1 and 2 were ovarian-derived. Ovariectomized ewes were implanted with progesterone-containing intravaginal implants and allotted to control (n = 5) or fasted (n = 5) treatment groups and fed as described for Trials 1 and 2. Similar to intact ewes, serum concentrations of progesterone were approximately twofold greater (P < 0.001) in fasted than in control implanted ovariectomized ewes. In summary, feed withdrawal for 5 d during the luteal phase of the estrous cycle increased serum concentrations of progesterone and evoked endocrine changes that could perturb the subsequent estrous cycle.


Subject(s)
Fasting/physiology , Growth Hormone/blood , Luteal Phase/physiology , Luteinizing Hormone/blood , Progesterone/blood , Sheep/physiology , Animals , Dinoprost/pharmacology , Drug Implants , Estradiol/blood , Fasting/blood , Female , Insulin/blood , Insulin-Like Growth Factor I/analysis , Luteal Phase/blood , Ovariectomy/veterinary , Pregnancy , Progesterone/administration & dosage , Random Allocation , Reproduction , Sheep/blood
4.
Reproduction ; 126(5): 615-20, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14611634

ABSTRACT

Cancer antigen 125 (CA-125) is expressed by malignant human ovarian surface epithelial cells and derivatives of the Müllerian duct system. This study explored the expression, regulation, and function of CA-125 in the bovine uterus. CA-125 was localized by immunohistochemistry to the apical surfaces of epithelial cells lining the endometrium and proximal glands of the late luteal phase and early pregnancy; antigen was not detected during oestrus or the postpartum period. Production of CA-125 by bovine endometrial cells in vitro was upregulated by progesterone and interferon-tau. Immunopurified CA-125 from uterine flushes of dioestrous or pregnant cows was similar in biochemical composition (as determined by gel electrophoresis and amino acid content) to the human antigen isolated from incubation medium conditioned by the ovarian cancer cell line OVCAR-3. Bovine CA-125 inhibited complement-induced lysis of antibody-sensitized sheep erythrocytes. It is suggested that endometrial CA-125 exerts a progestational role in part by protecting maternal and embryonic cells from immune targeting and lysis.


Subject(s)
CA-125 Antigen/analysis , Cattle/metabolism , Endometrium/chemistry , Estrus/metabolism , Postpartum Period/metabolism , Pregnancy, Animal/metabolism , Amino Acids/analysis , Animals , CA-125 Antigen/chemistry , CA-125 Antigen/metabolism , Cells, Cultured , Endometrium/metabolism , Estradiol/pharmacology , Female , Immunohistochemistry/methods , Interferon Type I/pharmacology , Pregnancy , Pregnancy Proteins/pharmacology , Progesterone/pharmacology
5.
Reproduction ; 124(3): 347-52, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12201808

ABSTRACT

Tissue dissolution and remodelling are associated with the processes of rupture of the ovulatory follicle and formation of the corpus luteum. Matrix metalloproteinase 2 (MMP-2) belongs to a family of endopeptidases that cleave extracellular proteins; its primary substrate is the lattice network of basement membranes that support epithelial cells and endothelium. The aim of this study was to ascertain a putative regulatory role of MMP-2 relevant to the folliculo-luteal transformation in ewes. Luteal regression and the preovulatory surge of gonadotrophins were synchronized by administration of PGF(2alpha) and GnRH on days 14.0 and 15.5 of the oestrous cycle, respectively. Dominant antral follicles present during pro-oestrus consistently ovulate approximately 24 h after GnRH administration. Normal IgG or a bioactivity-neutralizing MMP-2 monoclonal antibody was injected into the antral cavity of preovulatory follicles at 8 h after GnRH administration. Jugular blood samples were obtained for serum progesterone analysis and ovaries were removed for light microscopic morphometry on day 8. A definitive ovulation stigma was evident in control ewes. The antra of ruptured follicles had largely been supplanted with luteal tissue. In contrast, the ovarian surface contiguous with follicles injected with anti-MMP-2 was smooth and undisturbed, which is indicative of a failure of ovulation. Luteinized unruptured follicles were filled with (entrapped) fluid. Corpora lutea of control animals contained numerous connective tissue projections that provided a framework for cellular migration and angiogenesis. Luteal tissues that surrounded the cavity of antibody-treated follicles lacked trabeculae and were deficient in blood vessels. Systemic venous progesterone concentrations were lower in ewes with a luteinized unruptured follicle compared with those with a corpus luteum. It is proposed that MMP-2 is a mediator of ovulation and luteal development.


Subject(s)
Corpus Luteum/physiology , Matrix Metalloproteinase 2/physiology , Ovarian Follicle/physiology , Ovulation/physiology , Sheep/physiology , Animals , Antibodies, Monoclonal/immunology , Corpus Luteum/anatomy & histology , Female , Luteolysis/physiology , Matrix Metalloproteinase 2/immunology , Ovarian Follicle/anatomy & histology , Progesterone/blood
6.
Biol Reprod ; 65(5): 1417-24, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11673258

ABSTRACT

Oxidative base (8-oxoguanine) damage, DNA fragmentation, and apoptosis occurred among ovarian surface epithelial cells within the formative site of ovulation in sheep. The incidence of 8-oxoguanine adducts in surviving antiapoptotic Bcl-2/base excision repair polymerase beta-positive cells at the margins of ruptured follicles (which avoid the focal point of the ovulatory assault) was intermediate between apoptotic and outlying healthy epithelium. Cells containing perturbations to DNA expressed the tumor suppressor p53. Localized reactions of DNA injury and programmed cellular death were averted by ovulation blockade with indomethacin. Progesterone enhanced the biosynthesis of polymerase beta in ovarian surface epithelial cells exposed in vitro to a sublethal concentration of H(2)O(2). Ovulation is a putative etiological factor in common epithelial ovarian cancer. A genetically altered progenitor cell, with unrepaired DNA, but not committed to death, could give rise to a transformed phenotype that is hence propagated upon healing of the ovulatory wound; it appears that this incongruity is normally reconciled by up-regulation of the base excision repair pathway during the ensuing luteal phase.


Subject(s)
Apoptosis , DNA Damage , DNA Repair , Guanine/analogs & derivatives , Ovary/chemistry , Ovulation , Sheep , Animals , DNA Adducts , DNA Fragmentation , DNA Polymerase beta/analysis , DNA Polymerase beta/metabolism , Epithelial Cells/chemistry , Epithelial Cells/drug effects , Epithelial Cells/enzymology , Female , Gene Expression , Genes, p53/genetics , Guanine/analysis , Hydrogen Peroxide/pharmacology , Indomethacin/pharmacology , Ovarian Follicle/chemistry , Ovary/drug effects , Ovulation/drug effects , Progesterone/pharmacology , Proto-Oncogene Proteins c-bcl-2/analysis , Tumor Suppressor Protein p53/analysis
7.
J Steroid Biochem Mol Biol ; 78(2): 185-91, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11566443

ABSTRACT

Urokinase plasminogen activator (uPA) has been implicated in the metastatic potential of ovarian carcinomas of surface epithelial origin. The SKOV-3 human ovarian cancer cell line was tested for uPA secretory responses (enzyme immunoassay of conditioned media) after treatments with sex steroids, human menopausal gonadotropins (hMG), or gonadotropin-releasing hormone (GnRH). Secretion of uPA during a 6-h incubation was unaffected by testosterone, estradiol-17beta, hMG, or GnRH. Progesterone, at supraphysiological concentrations, suppressed uPA secretion; this reaction was not altered by the progesterone receptor antagonist RU486 or the transcriptional inhibitor actinomycin D. It appears that progesterone exerted a direct biophysical effect on the plasma membrane manifested by an interference with shedding of uPA in exocytotic vesicles. Finally, invasion of SKOV-3 cells into Matrigel was inhibited by progesterone. We suggest that progesterone can disrupt the fluid dynamics of plasma membranes and thereby invoke an antitumorigenic action via inhibition of proteolytic secretions.


Subject(s)
Adenocarcinoma/drug therapy , Cell Membrane/drug effects , Ovarian Neoplasms/drug therapy , Progesterone/pharmacology , Urokinase-Type Plasminogen Activator/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Estradiol/pharmacology , Female , Gonadotropin-Releasing Hormone/pharmacology , Gonadotropins/pharmacology , Humans , Mifepristone/pharmacology , Neoplasm Invasiveness/prevention & control , Receptors, Progesterone/antagonists & inhibitors , Testosterone/pharmacology , Tumor Cells, Cultured
8.
Mol Reprod Dev ; 58(4): 417-23, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11241778

ABSTRACT

The basic premise of this investigation was that local hormonal control of stockpiling of the base excision repair polymerase (poly) beta within oocytes of preovulatory follicles occurs as a function of cytoplasmic maturation. There was an increase in immunoreactive poly beta in sectioned oocytes of preovulatory ovine follicles during a 12-36-hour interval following the onset of prostaglandin (PG) F2alpha-induced (Day 14 of the estrous cycle) luteal regression; this response was not observed in subordinate (nonovulatory) follicles. Accumulation of poly beta in oocytes at 36 hr after PGF2alpha was negated by treatment of ewes at 12 hr with the aromatase inhibitor Arimidex or an ovulatory dose of GnRH (which, via surge gonadotropin stimulation, acutely downregulates the proestrous rise in follicular estrogen biosynthesis). Estradiol-17beta stimulated poly beta expression (transcriptional control) in oocytes of explanted (12 hr after PGF2alpha) follicles (24-hour incubation). We suggest that a critical period of estrogen amplification in the preovulatory follicle underscores the capacity of its oocyte to efficiently repair DNA and therefore reconcile spontaneous infidelities in genomic integrity that inevitably occur during preimplantation embryogenesis.


Subject(s)
DNA Polymerase beta/biosynthesis , DNA Repair/physiology , Estradiol/pharmacology , Oocytes/drug effects , Ovarian Follicle/drug effects , Sheep/physiology , Anastrozole , Animals , Aromatase Inhibitors , Blastocyst/metabolism , Corpus Luteum/drug effects , DNA Polymerase beta/genetics , Dactinomycin/pharmacology , Dinoprost/pharmacology , Drug Administration Schedule , Enzyme Induction/drug effects , Enzyme Inhibitors/pharmacology , Estrus , Female , Follicular Phase , Gonadotropins/metabolism , Meiosis , Nitriles/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Oocytes/enzymology , Ovarian Follicle/enzymology , Ovulation Induction , Triazoles/pharmacology
9.
J Anim Sci ; 78(9): 2237-43, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10985393

ABSTRACT

The effect of active immunization against GnRH on production, carcass, and behavioral traits was examined in ram lambs fed to a uniform slaughter weight. Lambs (initial BW = 32.6+/-1 kg) were stratified by BW and assigned at random to one of four treatment groups (n = 12 lambs/group). Lambs were untreated, castrated, or actively immunized against GnRH using a GnRH-keyhole limpet hemocyanin conjugate (1 mg) emulsified with either Freund's complete adjuvant (FCA) or another oil-based adjuvant (ISA). Animals were housed individually and slaughtered at 58 kg BW. Immunoneutralization of GnRH reduced (P < .05) testes weight and the concentration of testosterone in serum at slaughter. Suppression of testicular size and function was most clearly evident in animals immunized using FCA. Final anti-GnRH titer was also highest in lambs immunized using FCA. Several measures of sexual behavior (frequency of mounts and ejaculations) were also reduced (P < .05) in animals immunized using FCA. The duration of the feeding period was greater (P < .05) for castrated lambs than for untreated lambs, and intermediate feeding periods were required for FCA and ISA lambs. Average daily gain was greater (P < .05) in untreated than in castrated, FCA, or ISA lambs. Similarly, feed efficiency for untreated lambs was greater (P < .05) than for castrated, FCA, or ISA lambs, but feed efficiency did not differ among castrated, FCA, or ISA lambs. Longissimus muscle area, lean and bone maturity, overall quality, muscling score, flank streaking, and color of fat did not differ among treatments. Intact, FCA, and ISA lambs had more (P < .05) desirable yield grades, less (P < .05) backfat, and less (P < .05) marbling than castrated lambs. In summary, immunization against GnRH decreased testicular weight and reduced (P < .05) feedlot performance and sexual behavior to levels comparable to those of castrated males. Partitioning of nutrients for growth and deposition of fat, however, seems to differ among immunologically castrated and physically castrated lambs. This difference in nutrient partitioning may be due to residual testicular activity in immunized lambs.


Subject(s)
Gonadotropin-Releasing Hormone/immunology , Sexual Behavior, Animal/physiology , Sheep/growth & development , Testis/physiology , Vaccination/veterinary , Animal Feed , Animal Husbandry , Animals , Body Weight , Male , Testis/growth & development
10.
Biol Signals Recept ; 9(2): 102-14, 2000.
Article in English | MEDLINE | ID: mdl-10810205

ABSTRACT

Collagen breakdown and cellular death (apoptosis and inflammatory necrosis) within the apex of preovulatory ovine follicles are hallmarks of impending ovarian rupture. An integrative mechanism is presented whereby gonadotropic stimulation of urokinase-type plasminogen activator secretion by ovarian surface epithelial cells bordering the preovulatory follicle elicits a localized increase in tissue plasmin, which activates latent collagenases and secretion of tumor necrosis factor-alpha (TNF-alpha) from thecal endothelium. TNF-alpha potentiates collagenolysis (via matrix metalloproteinase gene expression) and (at elevated concentrations) mediates epithelial/vascular dissolution. Incidental damage to DNA of ovarian surface epithelial cells circumjacent to the ruptured follicle is a putative etiological factor in ovarian cancer.


Subject(s)
Ovary/physiology , Ovulation/physiology , Peptide Hydrolases/physiology , Animals , Cell Death/physiology , Female , Humans , Ovary/cytology
11.
Reprod Fertil Dev ; 12(1-2): 75-80, 2000.
Article in English | MEDLINE | ID: mdl-11194561

ABSTRACT

The collagenous matrix of the wall of periovulatory follicles is degraded and remodelled during ovulatory ovarian rupture and luteinization. Matrix metalloproteinase-2 (MMP-2) belongs to a family of zinc endopeptidases that cleave extracellular proteins; its primary substrate is the type IV collagen of basement membranes. Tumour necrosis factor alpha (TNFalpha) is a putative mediator of collagenolysis and ovulation. The objective of this investigation was to ascertain the regulatory role of TNFalpha on MMP-2 activity relevant to the folliculo-luteal transition in ewes. Luteal regression and the preovulatory surge of gonadotropins were induced by administration of prostaglandin F2alpha and gonadotropin-releasing hormone (GnRH) on Days 14 and 15.5 (= 0 h) of the oestrous cycle, respectively. Ovulation occurs from the dominant follicle approximately 24 h after GnRH. An immunocapture-activity assay was used to measure MMP-2 in follicular extracts. Bioactive MMP-2 increased from 0 to 20 to 40 h after GnRH. Enzyme was immunolocalized at 40 h to the connective tissue framework that invades the parenchyma of the formative corpus luteum. Activity of MMP-2 was up-regulated by incubation (20 h) of 0-h follicular explants with TNFalpha; this response was suppressed by the transcriptional inhibitor actinomycin D. Activity of MMP-2 was reduced when preovulatory follicular tissues were incubated (12-h explants for 6 h) with TNFalpha antiserum. Ovulation was blocked by intrafollicular injection of TNFalpha antiserum. Unruptured follicles luteinized, but were deficient in collagenous/vascularized trabeculae, and produced less progesterone than their control luteal counterparts. It is suggested that TNFalpha, via MMP-2 induction, contributes to the reorganization of an ovulatory follicle into a fully competent corpus luteum.


Subject(s)
Matrix Metalloproteinase 2/metabolism , Ovarian Follicle/enzymology , Ovulation , Sheep/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Collagen/metabolism , Corpus Luteum/drug effects , Corpus Luteum/metabolism , Dactinomycin/pharmacology , Dinoprost/pharmacology , Female , Gonadotropin-Releasing Hormone/pharmacology , Immune Sera/pharmacology , Luteolysis , Male , Nucleic Acid Synthesis Inhibitors/pharmacology , Ovarian Follicle/drug effects , Ovulation/drug effects , Tumor Necrosis Factor-alpha/immunology
12.
Reprod Fertil Dev ; 12(3-4): 127-32, 2000.
Article in English | MEDLINE | ID: mdl-11302421

ABSTRACT

Eosinophilic leukocytes infiltrate the wall of postovulatory ovine follicles. The objective of this investigation was to assess a putative role of resident eosinophils in the folliculo luteal transition. Eosinophils accumulated where new blood vessels were evident along connective tissue trabeculae that pervaded the parenchyma of formative corpora lutea. Mid-phase function of corpora lutea (progesterone output) was suppressed in ewes in which eosinophils were ablated by systemic administration of prednisolone following ovulation. Glandular dysfunction was related to a diminished angiogenic response (quantitative image analysis of vascular space in histological specimens and scanning electron microscopy of corrosion casts) during the luteinization process. Vascular endothelial growth factor (VEGF) was localized by immunofluorescence microscopy to luteal eosinophils. It is suggested that VEGF of eosinophilic origin contributes to the neovascularization mechanism of corpora lutea in sheep.


Subject(s)
Corpus Luteum/drug effects , Corpus Luteum/growth & development , Eosinophils/drug effects , Leukopenia/chemically induced , Leukopenia/physiopathology , Luteal Phase/drug effects , Luteal Phase/physiology , Prednisolone/pharmacology , Animals , Corpus Luteum/blood supply , Corpus Luteum/physiology , Corrosion Casting , Endothelial Growth Factors/physiology , Eosinophils/physiology , Female , Lymphokines/physiology , Microscopy, Electron, Scanning , Neovascularization, Physiologic/drug effects , Sheep , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
13.
Biol Reprod ; 61(6): 1487-91, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10569993

ABSTRACT

Secretion of urokinase plasminogen activator (uPA) by ovarian surface epithelium (OSE) adjacent to the preovulatory ovine follicle has been implicated in apical tissue degradation and follicular rupture. In vitro experiments were designed to test the hypothesis that uPA release by OSE is under direct hormonal control. Epithelial cells were isolated from the ovarian surface of sheep using a polytetrafluorethylene scraper designed to dislodge adherent cells from culture flasks. Amidolytic cleavage of a uPA-specific chromogen (carbobenzoxy-L-gamma-glutamyl [alpha-ot-but]-glycyl-arginine-p-nitroanilide monoacetate) was used as a measure of enzymatic bioactivity in OSE-conditioned incubation media. Secretion of uPA by OSE suspensions from proestrous ewes was stimulated by exposure (2 h) to a preovulatory surge-like concentration of LH. OSE cells obtained during the luteal phase or anestrus were not responsive to LH. Baseline rates of uPA secretion and expression of estradiol receptors (in situ immunofluorescence detection) were not affected by reproductive status. Induction of uPA secretion by anestrous OSE was attained after priming (6 h) with estradiol-17beta; responsiveness was attributed to gonadotropin receptor (ligand binding) up-regulation. Monolayers of OSE established on polyethylene membranes secreted uPA predominately in a basal (i.e., toward the substratum) direction. We suggest that OSE in juxtaposition with the (hyperemic) wall of the preovulatory follicle is perfused by surge levels of LH, invoking uPA release into underlying ovarian tissues.


Subject(s)
Ovary/enzymology , Sheep/physiology , Urokinase-Type Plasminogen Activator/metabolism , Anestrus , Animals , Culture Media, Conditioned , Epithelial Cells/enzymology , Estradiol/pharmacology , Estrogen Receptor alpha , Female , Follicular Phase , Luteal Phase , Luteinizing Hormone/pharmacology , Proestrus , Receptors, Estrogen/analysis
14.
Biol Reprod ; 61(6): 1581-5, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10570006

ABSTRACT

The pleiotropic cytokine tumor necrosis factor (TNF)-alpha has been implicated in the mechanism of ovulation. Experiments were designed to test the hypothesis that TNF-alpha secreted from the oocyte-cumulus cell complex stimulates follicular collagenase production and thereby contributes to ovarian wall degradation and ovulatory rupture. Proestrous ewes were treated with GnRH to synchronize the onset of the gonadotropin surge; ovulation occurs approximately 24 h later. There was an increase in TNF-alpha (immunoassay) in antral fluid of preovulatory follicles at 18 h after GnRH, which was related to tissue collagenolytic bioactivity (radiolabeled type I substrate digestion by enzymatic extract) and collagen (hydroxyproline) depletion. Intrafollicular injection of TNF-alpha antibodies at 12 h after GnRH negated the rise in follicular collagenolytic bioactivity (and is known to block ovulation in the sheep). Moreover, collagenase production was enhanced when follicular tissues (0 h GnRH) were incubated (6 h) with recombinant TNF-alpha; this effect was abolished by the transcriptional inhibitor actinomycin D. Secretion of TNF-alpha by oocyte-cumulus cell complexes isolated from preovulatory follicles simulated the in vivo circumstance. Immunostaining indicated that TNF-alpha was confined mainly to the oocyte before GnRH administration, accumulated in cumulus cells during the mid-to-late preovulatory period, and was expended with the imminent approach of ovulation. To our knowledge, this is the first report specifying that up-regulation of collagenase expression is a target mode of TNF-alpha action in preovulatory follicles. The oocyte-cumulus cell complex is an apparent source of soluble TNF-alpha.


Subject(s)
Collagenases/metabolism , Cytokines/metabolism , Ovarian Follicle/physiology , Ovulation , Sheep/physiology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Collagen/metabolism , Cytoplasm/chemistry , Female , Follicular Fluid/metabolism , Gonadotropin-Releasing Hormone/pharmacology , Oocytes/physiology , Oocytes/ultrastructure , Proestrus , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/metabolism
15.
Biol Reprod ; 61(6): 1655-9, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10570016

ABSTRACT

Indomethacin, a nonsteroidal anti-inflammatory agent, is a potent inhibitor of ovulation in vertebrates. The presumptive obligate anovulatory mode of indomethacin action is via suppression of ovarian prostaglandin production. We report that a very high systemic dose of indomethacin (800 mg i.m.) is required to block ovulation in gonadotropin-treated anestrous ewes. A lower dose of indomethacin (200 mg), which negated the preovulatory rise in follicular prostaglandin (PGF(2alpha)) biosynthesis, did not prevent ovulation. Endothelial secretion of tumor necrosis factor (TNF)-alpha within the apical follicular wall (prospective site of rupture) was not altered by indomethacin; notwithstanding, the apoptosis (DNA-fragmentation)-inducing effect of TNF-alpha (a determinant of ovulatory stigma formation) was attenuated by 800 (but not 200) mg indomethacin. A suprapharmacological concentration of indomethacin also was necessary to protect ovarian surface epithelial cells from a (prostaglandin-independent) cytotoxic effect of TNF-alpha in vitro. It is concluded that indomethacin inhibits ovulation by anti-apoptotic mechanisms that can be dissociated from the paradigm of prostanoid down-regulation.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/drug effects , Indomethacin/pharmacology , Ovary/cytology , Ovulation/drug effects , Sheep/physiology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , DNA Fragmentation , Dinoprost/biosynthesis , Epithelial Cells/physiology , Female , Indomethacin/administration & dosage , Ovarian Follicle/drug effects , Ovarian Follicle/metabolism
16.
Endocrine ; 10(3): 243-52, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10484288

ABSTRACT

The interferon stimulated gene product, ISG17, conjugates to bovine uterine proteins in response to conceptus-derived interferon (IFN)-tau. The objectives of the present experiments were to examine induction of ISG17 (0.65 kb) and a related 2.5 kb mRNA in response to IFN-tau and pregnancy using Northern blotting procedures, and to determine cell types in the endometrium that expressed ISG17 mRNA using in situ hybridization. RNA was isolated from endometrial explants or from bovine endometrial (BEND) cells cultured in the absence (control) or presence of 25 nM recombinant (r) bolFN-tau for 0, 3, 6, 12, 24, or 48 h. The major ISG17 0.65 kb mRNA and a minor 2.5 kb mRNA were induced (p<0.05) after 6 h (explants) or 3 h (BEND cells) treatment with rboIFN-tau. Both mRNAs were present in endometrium from day 18 pregnant cows, but were absent in endometrium from nonpregnant cows. The ISG17 mRNA was localized to stromal and glandular epithelial cells on d 18 of pregnancy. The 2.5 kb mRNA may encode a novel ISG17 homolog, or a unique polyISG17 repeat that is similar in structure to the polyubiquitin genes. Because ISG17 mRNA is induced in stromal and glandular epithelial cells, it could be assumed that ISG17 has a role in regulating intracellular proteins in both cell types.


Subject(s)
Endometrium/metabolism , Interferon Type I/pharmacology , Nuclear Proteins/genetics , Pregnancy Proteins/pharmacology , RNA, Messenger/biosynthesis , Animals , Blotting, Northern , Cattle , Cell Line , Endometrium/chemistry , Epithelial Cells/chemistry , Female , In Situ Hybridization , Pregnancy , RNA, Messenger/analysis , Recombinant Proteins/pharmacology , Stromal Cells/chemistry , Tissue Distribution
17.
Biol Reprod ; 61(2): 388-92, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10411516

ABSTRACT

The objectives of this investigation were to determine the intrafollicular mechanisms and physiological consequences of estradiol actions in preovulatory ovine follicles. Acute suppression of estradiol production in proestrous ewes by an aromatase inhibitor (Arimidex) was associated with follicular lipid peroxidation, testosterone accumulation, and a granulosa cell deficiency (decreased proliferation/increased apoptosis). Estradiol-17beta stimulated granulosa proliferating cell nuclear antigen (PCNA) and protected cells from oxidative (H(2)O(2)) stress-induced apoptosis in vitro; the PCNA, but not the antiapoptotic response, was negated by the transcriptional inhibitor actinomycin D. Thus, it appears that genomic/mitotic and cytoprotective (oxygen-scavenging) modes of estradiol action operate in preovulatory follicles. Luteal (large steroidogenic cell) function was diminished following ovulation induction of estradiol-deficient follicles. It is suggested that inadequate exposure of the preovulatory follicle to estradiol caused the granulosa lutein insufficiency.


Subject(s)
Antioxidants/metabolism , Corpus Luteum/drug effects , Estradiol/pharmacology , Mitosis/drug effects , Ovarian Follicle/drug effects , Anastrozole , Animals , Apoptosis , Aromatase Inhibitors , Corpus Luteum/metabolism , DNA Fragmentation , Enzyme Inhibitors/pharmacology , Female , Nitriles/pharmacology , Ovarian Follicle/metabolism , Oxidative Stress , Progesterone/blood , Proliferating Cell Nuclear Antigen/metabolism , Sheep , Triazoles/pharmacology
18.
Biol Reprod ; 60(5): 1166-71, 1999 May.
Article in English | MEDLINE | ID: mdl-10208979

ABSTRACT

Ovulation in the sheep is predicated on plasmin up-regulation at the ovarian surface-follicular interface, release of tumor necrosis factor (TNF) alpha from contiguous endothelium, and apoptotic cell death. The objectives of this investigation were to determine whether plasmin elicits TNFalpha secretion from thecal endothelium of ovine follicles, to characterize the site(s) of enzymatic attack, and to assess the physiological consequence of soluble TNFalpha action. Endothelial cells of thecal tissues isolated from antral follicles of eCG-primed anestrous ewes shed (histochemical depletion) TNFalpha into incubation medium (ovarian cell DNA fragmentation bioassay, Western blot detection) upon exposure to plasmin. Immunopurification and N-terminal sequence analysis indicated that TNFalpha was excised from its transmembrane precursor at the Arg79-Ser80 and Lys88-Pro89 linkages. Microinjection of TNFalpha into the apical wall of explanted follicles induced cellular apoptosis and stigma development. We suggest that plasmin-mediated cleavage of TNFalpha exodomain from its membrane anchor along thecal endothelium is a determinant of tissue dissolution within the formative ovulatory rupture site of ewes.


Subject(s)
Fibrinolysin/metabolism , Ovarian Follicle/metabolism , Ovulation/physiology , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , DNA Fragmentation , Endothelium/cytology , Endothelium/drug effects , Endothelium/metabolism , Female , Granulosa Cells/drug effects , Granulosa Cells/metabolism , Immunohistochemistry , Microinjections , Ovarian Follicle/cytology , Ovarian Follicle/drug effects , Sheep , Theca Cells/drug effects , Theca Cells/metabolism , Tumor Necrosis Factor-alpha/pharmacology
19.
J Reprod Fertil Suppl ; 54: 353-8, 1999.
Article in English | MEDLINE | ID: mdl-10692867

ABSTRACT

Collagen breakdown and apoptotic cell death within the apex of the preovulatory ovine follicle are hallmarks of impending ovarian rupture. An integrative mechanism is proposed whereby gonadotrophic stimulation of urokinase-type plasminogen activator secretion by the follicular-contiguous ovarian surface epithelium elicits a localized increase in tissue plasmin, which activates collagenolysis and tumour necrosis factor alpha-induced cell death within the formative ovulatory stigma.


Subject(s)
Fibrinolysin/metabolism , Ovulation/physiology , Sheep/physiology , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis , Collagen/metabolism , Female , Ovarian Follicle/physiology
20.
J Reprod Fertil ; 117(2): 325-9, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10690200

ABSTRACT

The aim of this study was to define the temporal and spatial patterns of apoptosis, necrosis and inflammation within preovulatory ovine follicles. A gonadotrophin surge was induced in pro-oestrous ewes by GnRH, and isolated follicles were hemisected into apical and basal segments at 0, 10, 18 and 22 h (the time of ovulatory stigma development) after GnRH. Ovarian surface epithelial and granulosa cells were isolated and assessed by fluorescence microscopy for membrane phosphatidylserine translocation-annexin V (early-stage apoptosis), oligonucleosomal DNA nick endlabelling (advanced apoptosis), and nuclear propidium iodide incorporation (necrotic membrane disruption). Thecal shells were analysed for interstitial blood cells. Preovulatory follicles were also hemisected and subjected to electrophoretic DNA degradation analysis. Annexin V binding and in situ DNA fragmentation among ovarian surface epithelial and granulosa cells along the follicular apex were high 18 and 22 h after GnRH. Propidium iodide staining of apical ovarian surface and granulosa cells was apparent at 22 h. There was a coincident increase within the apical theca as the time of ovulation approached in extravasated leucocytes (18 and 22 h) and erythrocytes (22 h). Apoptotic DNA laddering and necrotic DNA smears within the follicular apex were evident on agarose gels at 18 and 22 h, respectively. In contrast, ovarian surface epithelium not associated with the ovulation site and the basal follicular wall were largely unafflicted. It is suggested that both modalities of cellular death, apoptosis and necrosis (with acute inflammation and vascular injury), contribute progressively to follicular stigma formation and ovarian rupture.


Subject(s)
Apoptosis , Ovarian Follicle/physiology , Ovulation/physiology , Sheep/physiology , Animals , Culture Techniques , DNA Fragmentation , Erythrocytes/physiology , Female , Gonadotropin-Releasing Hormone/pharmacology , Inflammation , Leukocytes/physiology , Necrosis , Ovarian Follicle/immunology , Ovarian Follicle/pathology , Ovulation Induction
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