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.

Increased expression of GPI-specific phospholipase D in mouse models of type 1 diabetes.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a high-density lipoprotein-associated protein. However, the tissue source(s) for circulating GPI-PLD and whether serum levels are regulated are unknown. Because the diabetic state alters lipoprotein metabolism, and liver and pancreatic islets are possible sources of GPI-PLD, we hypothesized that GPI-PLD levels would be altered in diabetes. GPI-PLD serum activity and liver mRNA were examined in two mouse models of type 1 diabetes, a nonobese diabetic (NOD) mouse model and low-dose streptozotocin-induced diabetes in CD-1 mice. With the onset of hyperglycemia (2- to 5-fold increase over nondiabetic levels), GPI-PLD serum activity and liver mRNA increased 2- to 4-fold in both models. Conversely, islet expression of GPI-PLD was absent as determined by immunofluorescence. Insulin may regulate GPI-PLD expression, because insulin treatment of diabetic NOD mice corrected the hyperglycemia along with reducing serum GPI-PLD activity and liver mRNA. Our data demonstrate that serum GPI-PLD levels are altered in the diabetic state and are consistent with liver as a contributor to circulating GPI-PLD.

Iron overload diminishes atherosclerosis in apoE-deficient mice.

It has been proposed that elevated levels of tissue iron increase the risk for atherosclerosis, perhaps by favoring the formation of pro-atherogenic oxidized LDL. Working with apoE-deficient (apoE(-/-)) mice, which do not require a high-fat diet to develop atherosclerosis, we compared the effects of standard diet (0.02% iron) or a 2% carbonyl iron diet. After 24 weeks, mice fed the 2% carbonyl iron diet had twice as much iron in their plasma, a ninefold increase in bleomycin-detectable free iron in their plasma, and ten times as much iron in their livers as control mice. Dietary iron overload caused a modest (30%) rise in plasma triglyceride and cholesterol. Nevertheless, this regimen did not exacerbate, but rather reduced the severity of atherosclerosis by 50%, and it failed to elevate hepatic levels of heme oxygenase mRNA, which is induced by many different oxidative insults in vitro. Moreover, hepatic levels of protein-bound dityrosine and ortho-tyrosine, two markers of metal-catalyzed oxidative damage in vitro, failed to rise in iron-overloaded animals. Our observations suggest that elevated serum and tissue levels of iron are not atherogenic in apoE(-/-) mice. Moreover, they call into question the hypothesis that elevated levels of tissue iron promote LDL oxidation and oxidative stress in vivo.

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.

Impaired superoxide production due to a deficiency in phagocyte NADPH oxidase fails to inhibit atherosclerosis in mice.

Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase-a model of chronic granulomatous disease (CGD)-to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E-deficient (apoE-/-) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE-/- animals had lower plasma cholesterol and triglyceride levels than did the apoE-/- animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.

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.

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.

Initiation of hyperinsulinemia and hyperleptinemia is diet dependent in C57BL/6 mice.

C57BL/6 female mice were fed high fat diets containing different types of carbohydrate (sucrose or corn starch) and contents of cholesterol (0.03 % or 1 %) to identify early metabolic changes leading to increases in leptin levels and eventual insulin resistance. Under identical dietary fat conditions, type of carbohydrate and cholesterol content contributed to the timing of leptin increases. Mice fed a high-fat, high-sucrose diet showed early (4 weeks) and robust increases in circulating insulin and leptin levels (2-fold and 5-fold, respectively). In contrast, mice fed this diet with added cholesterol or with sucrose substituted by corn starch led to marked delays (8-10 weeks) in the elevations of insulin and leptin, although body weight gains were nearly identical among test diet groups. Thus, sucrose in combination with saturated fat played a specific role in initiating early metabolic changes associated with elevated leptin and insulin levels. Because leptin levels were most reflective of changes in insulin, our data support a role for insulin in determining plasma leptin levels in mice.

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.

Dietary antioxidants inhibit development of fatty streak lesions in the LDL receptor-deficient mouse.

Oxidized low density lipoprotein (LDL) promotes atherogenesis. Although pharmacological antioxidants such as probucol inhibit both LDL oxidation and atherosclerosis in hyperlipidemic animals, the effects of natural antioxidants such as vitamin E are inconclusive. To further determine the effects of supplemental dietary antioxidants in vivo, we evaluated whether combined dietary antioxidants (0.1% vitamin E, 0.5% beta-carotene, and 0.05% vitamin C) inhibit LDL oxidation and fatty streak lesion development in homozygous LDL receptor-null (LDLR-/-) mice fed a high-fat, high-cholesterol diet. An additional group of mice were fed black tea, which has been shown to inhibit LDL oxidation in vitro. After receiving a high-fat, high-cholesterol diet for 8 weeks, the combined antioxidant-supplemented (antioxidant) group (n=18), tea group (n=19), and control group (n=17) had equivalent plasma cholesterol levels. LDL oxidation, as measured by the lag phase of conjugated diene formation, was markedly inhibited in the antioxidant group compared with the tea or control groups [mean lag phases=143+/-7 (antioxidant), 100+/-5 (tea), and 84+/-4 (control) minutes; P<0.0001 antioxidant versus tea or control]. The cross-sectional surface area of fatty streak lesions in the aortic sinus was reduced by 60% in the antioxidant group compared with both the tea and control groups (P<0.0001 antioxidant versus tea or control). There was no difference in lesion area between tea and control groups. Although both LDL oxidation and atherosclerosis were significantly inhibited in the antioxidant group, no correlation between lag phase values and lesion size was observed among individual animals. Furthermore, black tea did not inhibit fatty streak development in LDLR-/- mice. These data suggest that combined natural dietary antioxidants inhibit both LDL oxidation and atherogenesis in animals with elevated LDL but that inhibition of LDL oxidation alone may not prevent the development of atherosclerosis.

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.

Dietary isoflavones reduce plasma cholesterol and atherosclerosis in C57BL/6 mice but not LDL receptor-deficient mice.

Susceptibility to atherosclerosis is determined by a combination of genetic and environmental factors, including diet. Consumption of diets rich in soy protein has been claimed to protect against the development of atherosclerosis. Potential mechanisms include cholesterol lowering, inhibition of lipoprotein oxidation and inhibition of cell proliferation by soy proteins or isoflavones, such as genistein, that are present in soy. This study was designed to determine whether soy isoflavones confer protection against atherosclerosis in mice and whether they reduce serum cholesterol levels and lipoprotein oxidation. C57BL/6 and LDL receptor-deficient (LDLr-null) mice were fed soy protein-based, high fat diets with isoflavones present (IF+, 20.85 g/100 g protein, 0.027 g/100 g genistein, 0.009 g/100 g daidzein) or diets from which isoflavones, and possibly other components, had been extracted (IF-, 20.0 g/100 g protein, 0.002 g/100 g genistein, 0.001 g/100 g daidzein). Because LDLr-null mice develop extensive atherosclerosis and hypercholesterolemia after minimal time on a high fat diet, they were fed the diets for 6 wk, whereas C57BL/6 mice were fed the diets for 10 wk. Plasma cholesterol levels did not differ between LDLr-null mice fed IF- and those fed IF+, but were 30% lower in C57BL/6 mice fed the IF+ diet than in those fed the IF- diet. Susceptibility of LDL to oxidative modification, measured as the lag phase of conjugated diene formation in LDLr-null mice, was not altered by isoflavone consumption. All LDLr-null mice developed atherosclerosis, and the presence or deficiency of dietary isoflavones did not influence atherosclerotic lesion area. In contrast, atherosclerotic lesion area was significantly reduced in C57BL/6 mice fed IF+ compared with those fed IF-. Thus, this study demonstrates that although the isoflavone-containing diet resulted in a reduction in cholesterol levels in C57BL/6 mice, it had no effect on cholesterol levels or on susceptibility of LDL to oxidative modification in LDLr-null mice. Further, dietary isoflavones did not protect against the development of atherosclerosis in LDLr-null mice but did decrease atherosclerosis in C57BL/6 mice. These findings suggest that soy isoflavones might lower cholesterol levels by increasing LDL receptor activity, and the reduction in cholesterol may offer some protection against atherosclerosis.