Mechanisms and pathobiology of ovulation.

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.

Effects of feeding high-linoleate safflower seeds on postpartum reproduction in beef cows.

Two experiments were conducted to evaluate reproductive responses to supplemental high-linoleate safflower seeds in postpartum beef cows. In Exp. 1, 18 primiparous, crossbred beef cows (411 +/- 24.3 kg of BW) were fed Foxtail millet hay starting 1 d postpartum at 1.68% of BW (DM basis) and a low-fat control (control: 63.7% cracked corn, 33.4% safflower seed meal, and 2.9% liquid molasses; DM basis) at 0.35% of BW (n = 9) or a supplement (linoleate) containing 95.3% cracked high-linoleate (79% 18:2n-6) safflower seeds and 4.7% liquid molasses (DM basis) at 0.23% of BW (n = 9). Beginning 1 d postpartum, blood was collected every 3 d for sera. Cows were slaughtered at 37 +/- 3 d postpartum for collection of hypothalami, anterior pituitary glands, liver, ovarian follicles, and uterine tissue. By 37 +/- 3 d postpartum, dietary treatment did not influence ovarian follicular development (P >or= 0.17), hypophyseal concentrations of LH (P = 0.14), or concentrations of IGF-I in liver (P = 0.15). In contrast, anterior pituitary glands from linoleate cows contained more FSH (P = 0.02) than control cows and linoleate cows had less IGF-I in the medial basal hypothalamus (P = 0.05), preoptic area (P = 0.06), and in follicular fluid (P

Influence of short-term fasting during the luteal phase of the estrous cycle on ovarian follicular development during the ensuing proestrus of ewes.

Short-term fasting of mature ewes during diestrus results in increased serum concentrations of progesterone and a delayed pre-ovulatory surge release of LH. To determine if these changes in reproductive hormones influence subsequent follicular development, mature ewes observed in estrus were assigned randomly to control (n=10) or fasted (n=15) groups. Control ewes had ad libitum access to feed, whereas fasted ewes were not fed from day 7 through 11 of their estrous cycle. Daily blood samples were collected from control and fasted ewes throughout the fasting period. Fasting increased (P<0.001) serum concentrations of progesterone (4.4 ng/mL versus 2.7 ng/mL [+/-0.3]). On day 12, all ewes were treated with 10mg of PGF(2alpha) and fasted ewes were returned to ad libitum feed. Ovaries were collected from ewes (n=5 each group) at 0 and 72 h following PGF(2alpha) in control and 0, 72 and 96 h in fasted ewes. Ovaries were weighed and small (< or =2mm), medium (3-4mm), and large (> or =5mm) follicles were enumerated. Total numbers of follicles were less (P<0.001) in fasted than fed ewes (14.6 versus 30.2 [+/-2.2]) at 0 h, but did not differ (P=0.9) when numbers of follicles were compared at similar times before the anticipated LH surge (i.e., at 72 h versus 96 h in control and fasted ewes, respectively). Within follicular size class, numbers of small and medium follicles were decreased (P=0.04) at 0 h in fasted ewes. Numbers of large follicles did not differ (P=1.0) between groups. Although numbers of small and medium ovarian follicles in fasted ewes recovered by 96 h to values comparable to fed ewes at 72 h following PGF(2alpha), serum concentrations of estradiol 17beta (P=0.08) and FSH (P=0.06) tended to be decreased in fasted ewes before the anticipated surge release of LH. Pituitary content of LH and FSH also tended to be lower (P< or =0.09) at 96 h in fasted ewes than at 72 h in control ewes, but did not differ (P> or =0.4) at hour 0 following PGF(2alpha). Hypothalamic and stalk median eminence contents of GnRH were not influenced (P> or =0.2) by fasting at any time period. Fasting during the luteal phase perturbs gonadotropin secretion and may influence fertility by causing a delay in ovarian follicle development.

Effects of feed restriction on reproductive and metabolic hormones in ewes.

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.

Expression of CA-125 by progestational bovine endometrium: prospective regulation and function.

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.

Role of matrix metalloproteinase 2 in the ovulatory folliculo-luteal transition of ewes.

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.

Estrogenic upregulation of DNA polymerase beta in oocytes of preovulatory ovine follicles.

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.

Tumour necrosis factor alpha up-regulates matrix metalloproteinase-2 activity in periovulatory ovine follicles: metamorphic and endocrine implications.

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.

Aetiology of attenuated luteal development in prednisolone-induced eosinopenic ewes.

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.

Hormonal control of urokinase plasminogen activator secretion by sheep ovarian surface epithelial cells.

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.

Tumor necrosis factor alpha regulates collagenolytic activity in preovulatory ovine follicles: relationship to cytokine secretion by the oocyte-cumulus cell complex.

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.

Mitogenic and antioxidant mechanisms of estradiol action in preovulatory ovine follicles: relevance to luteal function.

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.

Plasmin cleaves tumor necrosis factor alpha exodomain from sheep follicular endothelium: implication in the ovulatory process.

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.

Luteal dysfunction in ewes induced to ovulate early in the follicular phase.

The purpose of this investigation was to determine whether the timing of ovulation induction during the follicular phase is a determinant of consequent luteal function. Ewes were treated on day 14 of the estrous cycle with PGF2alpha to synchronize luteal regression and 12 or 36 h later with an ovulatory dose of GnRH. Luteal phase serum progesterone concentrations of normal magnitude were characteristic of animals elicited to ovulate by GnRH injection 36 h after PGF2alpha treatment. Follicles stimulated at 12 h of the induced follicular phase formed subfunctional corpora lutea that were deficient in large steroidogenic cells. Endometrial gland development was attenuated in ewes exhibiting luteal insufficiency. The pathophysiology of the luteal defect was associated with a retrospective lack of granulosal cells in preovulatory follicles not adequately primed by estradiol. Preovulatory LH surges were not affected by the time of GnRH treatment. Corpus luteum rescue indicative of maternal recognition of pregnancy occurred in inseminated ewes that were injected with GnRH 36 h after PGF2alpha. Gonadotropic stimulation 12 h after PGF2alpha typically resulted in gestational failure; a marginal improvement in the pregnancy rate was attained by progesterone supplementation. We suggest that premature induction of ovulation compromises the estrogen-mediated succession of granulosal cell proliferative events that necessitate the formation of a fully competent corpus luteum.

Pregnancy and interferon-tau induce conjugation of bovine ubiquitin cross-reactive protein to cytosolic uterine proteins.

Conceptus-derived interferon-tau (IFN-tau) induces bovine endometrial ubiquitin cross-reactive protein (UCRP) mRNA and protein on Days 15-21 of pregnancy. Bovine UCRP retains the Leu-Arg-Gly-Gly C-terminal sequence of ubiquitin that ligates to and directs degradation of cytosolic proteins. The objectives of the present experiments were to determine whether UCRP became conjugated to endometrial cytosolic proteins during early pregnancy and in response to recombinant bovine (rbo) IFN-tau. Ubiquitin (8 kDa), UCRP (17 kDa), and conjugates thereof (> or = 30 kDa) were quantitated using Western blotting and densitometry. Endometrial ubiquitin and its conjugates did not differ between Day 18 pregnant and nonpregnant cows, or between control and rboIFN-tau-treated (25 nM) explant cultures (Day 14; nonpregnant). Bovine UCRP was induced in endometrium from pregnant as compared with nonpregnant cows. Conjugation of endometrial proteins to UCRP was induced in pregnant as compared to nonpregnant cows. Recombinant boIFN-tau induced UCRP and its conjugates in cultured endometrial explants from nonpregnant cows. It is concluded that UCRP, in response to rboIFN-tau, becomes conjugated to endometrial cytosolic proteins during early pregnancy. The regulation of uterine proteins by UCRP may be integral to the maintenance of early pregnancy in ruminants.

Inhibition by tocopherol of prostaglandin-induced apoptosis in ovine corpora lutea.

Accumulation of toxic oxidants within corpora lutea is a prelude of apoptotic cell death. Vitamin E (alpha-tocopherol) is a biological antioxidant that protects cells from the inductive effects of reactive oxygen on DNA damage and nuclear/cytoplasmic condensation that dictate apoptosis. Ewes were challenged with a luteolytic dose of PGF2 alpha on d 10 of the estrous cycle. The acute decline in circulatory progesterone indicative of the onset of functional luteolysis was not affected by systemic administration of alpha-tocopherol; however, corpora lutea consequently (beyond 24 h) rebounded from the steroidogenic insult. Luteal tissues obtained at 24 h after PGF2 alpha revealed that internucleosomal DNA fragmentation and cellular collapse were inhibited by alpha-tocopherol. These observations indicate that regressive corpora lutea can be spared from terminal involution by diminishing the apoptotic influence of luteolytic hormone with an antioxidant.

Annual cycle of plasma testosterone in male copperheads, Agkistrodon contortrix (Serpentes, Viperidae): relationship to timing of spermatogenesis, mating, and agonistic behavior.

Reproductive cycles of the majority of squamate reptiles remain undescribed. Few studies are available on seasonal patterns of circulating steroid hormones in snakes. The goal of this study was to document the annual cycle of plasma testosterone (T) in male copperheads Agkistrodon contortrix, a North American pitviper (Serpentes, Viperidae). Two experimental conditions were used in this laboratory study. One condition (repeat-test group) consisted of 10 adult males that were sampled once each month for 11 months. The other condition (single-test groups) consisted of 10 groups each with 5 males (N = 50), and each male was tested a single time. The single-test condition was used to evaluate whether or not repeated handling and sampling affected T levels. The study was conducted from February-December, 1992. A well-defined seasonal pattern of plasma T levels was detected; patterns were similar under both experimental conditions with the exception that the repeat-test group had slightly lower levels. Levels of T were lowest (baseline) in April-May, increased in early summer (June), and were highest in late summer (August). Thereafter, T levels declined up to the time of hibernation (early November) and changed little during hibernation (November-January). Upon emergence from hibernation in late winter (February), T levels increased sharply from February to March and then decreased from March to April. The results are discussed in the context of timing of spermatogenesis, mating, and male agonistic behavior.

Oestradiol inhibits spontaneous and cisplatin-induced apoptosis in epithelial ovarian cancer cells: relationship to DNA repair capacity.

A prospective role of sex steroid hormones in the pathogenesis of common epithelial ovarian cancer remains equivocal. We hypothesized that oestradiol can protect ovarian cells from apoptosis by augmenting their DNA repair capacity. Two established oestrogen receptor-positive human cancer cell lines of ovarian surface epithelial origin (OVCAR-3, SKOV-3) were studied during short-term (24 h) subculture in the absence or presence of oestradiol-17beta and/or the DNA-damaging chemotherapeutic agent cisplatin. Apoptosis was monitored among individual cells by in situ DNA fragmentation analysis. Basal rates of apoptosis were diminished by exposure to oestradiol (progesterone or testosterone were without effect). Oestradiol also suppressed apoptosis induced by cisplatin and enhanced the repair of a cisplatin-damaged reporter chloramphenicol-O-acetyltransferase gene transfected into ovarian cells. The ability of oestrogen-responsive ovarian cancer cells to efficiently repair DNA and thereby avoid apoptosis may be related to propensity for clonal expansion and drug resistance.

Effects of porcine relaxin on induced parturition in beef heifers.

Two-year-old crossbred beef heifers were used to test the effects of porcine relaxin (pRelaxin) alone, or in combination with dexamethasone, on the induction of parturition, the incidence of dystocia, and retained placentas. Effects of treatment on pelvic area, postpartum interval, milk production, colostrum quality, calf birth weight, calf vigor, and calf performance were also evaluated. On Day 275 of gestation, heifers from two fetal-sire groups were randomly assigned to one of four groups in a 2 x 2 factorial design and received; no treatment (controls, n = 19), 20 mg of dexamethasone intramuscularly (im) (n = 22), 5 mg of pRelaxin (3,000 U/mg) im (n = 19), or 20 mg of dexamethasone plus 5 mg of pRelaxin (n = 17). Length of gestation (in days) was less (P < 0.05) in heifers treated with dexamethasone (279.8 +/- 1.0) than in controls (286.6 +/- 0.9), but was not influenced (P > 0.05) by treatment with pRelaxin. The incidence of retained placentas in heifers treated only with dexamethasone (27.3%) was not reduced by concomitant treatment with pRelaxin (35.3%). Retained placentas were not observed in any control heifers and in only one heifer (5.2%) treated solely with pRelaxin. Ease of calving (1 = unassisted, 5 = abnormal presentation) was not influenced by treatment (P > 0.05), even though birth weights (in kilograms) of calves from heifers treated with dexamethasone (36.4 +/- 0.8) were less (P < .01) than those of calves from nondexa-methasone-treated heifers (39.2 +/- 0.8). Dexamethasone tended to reduce (P < 0.07) calf vigor (1 = healthy and strong, 5 = dead on arrival; 1.48 +/- 0.11 vs. 1.18 +/- 0.11), but was not (P > 0.05) influenced by pRelaxin. The duration of the postpartum anestrous interval (73.1 +/- 1.8 d across groups) and pelvic areas following treatment and parturition were not influenced (P > 0.05) by dexamethasone or pRelaxin. Although determinants of colostrum quality (P < 0.01) and quantity (P < 0.08) of milk produced were influenced by dexamethasone, adjusted 205-d weights of calves did not differ (P > 0.05) among groups. In conclusion, treatment with pRelaxin alone failed to induce parturition or, when combined with dexamethasone, to reduce the incidence of retained placentas.

Levels of plasma corticosterone and testosterone in male copperheads (Agkistrodon contortrix) following staged fights.

Fighting behavior between male copperheads (Agkistrodon contortrix) occurs during the two mating periods (late summer/fall and spring) to gain priority of access to females. Fights are characterized by prominent vertical challenge displays, swaying, and a high degree of physical contact that does not involve biting. At the moment of subordination, losers retreat quickly from fights and winners respond by chasing. Subsequently, losers do not participate in further challenge displays or fighting for at least 7 days, and also they show behavioral signs of stress, which includes submissive acts and suppression of sexual behavior. The goal of this study was to determine whether or not losers show elevated levels of plasma corticosterone (B) and depressed levels of plasma testosterone (T) relative to winners and controls. Winners and losers were produced in 13 staged trials. Two different controls (N = 26) were run. Males with no recent agonistic experience were (1) tested in the fighting arena in the absence of a competitive male but paired with a single female (N = 13), and (2) tested alone in their cages (N = 13). All trials, including controls, were conducted in spring and late summer. Mean B in losers at 1-hr postfight was significantly greater than in winners and both control groups in both seasons. Mean T was significantly greater in late summer in all groups, as expected, but in each season was not significantly different between the groups. Levels of B and T were not correlated with SVL, mass, or duration of fighting. This study provides further support for the social insensitivity/challenge hypotheses and is the first to document postfight B and T levels in snakes.