Progesterone production by cultured luteal cells in the presence of bovine low- and high-density lipoproteins purified by heparin affinity chromatography.

The objectives of this study were to separate plasma lipoprotein particles based on the presence (low-density lipoproteins; LDL) or absence of apolipoprotein B (high-density lipoproteins; HDL) and to compare the abilities of bovine LDL and HDL to stimulate progesterone production by bovine luteal cells in culture. Plasma lipoproteins were isolated by ultracentrifugation and separated into LDL and HDL by heparin affinity chromatography. Luteal cultures were treated with LDL or HDL cholesterol for 48 h on d 3 of the culture (d 0 = day of dissociation). Progesterone production by luteal cells increased in a dose-dependent manner with increasing concentrations of either LDL or HDL cholesterol. There were no differences in the ability of LDL or HDL cholesterol to stimulate luteal cells to produce progesterone. Because LDL and HDL were equally potent, and experiment was designed to investigate the ability of modified LDL or reconstituted particles without apolipoproteins to stimulate progesterone production. Stimulation of luteal cell progesterone production by lysine-modified LDL was 70% of unmodified LDL. Progesterone production in the presence of phosphatidylcholine liposomes or BSA containing cholesterol was 50 and 108% of that obtained with HDL or LDL. Evidence indicated that apolipoprotein-free particles that contained free cholesterol but not cholesterol esters stimulated luteal cells to produce progesterone.     

Corpus luteum function following spontaneous or prostaglandin-induced estrus in Brahman cows and heifers

Corpus luteum (CL) function following spontaneous or prostaglandin F2 alpha-induced estrus was studied in 27 Brahman cows and 16 Brahman heifers. Females received one injection of 0, .38, .75 or 2.25 mg alfaprostol (PGF)/100 kg body weight at 12 +/- .1 d post-estrus. Serum progesterone (P4) during CL formation, following the subsequent estrus, increased over time (P less than .001, 1 to 13 d post-estrus) and was influenced by PGF (P less than .06) and a PGF X time interaction (P less than .02). Low serum P4 concentrations were observed during CL formation (d 3, 4, 10, and 12) in cattle that received 2.25 mg PGF. On d 13 post-estrus, CL were removed. No differences in weight, P4 content or number of luteinizing hormone (LH) receptors was evident between CL derived 13 d following spontaneous vs PGF-induced estrus. The CL formed following PGF-induced estrus had fewer large (P less than .002) and small (P less than .09) luteal cells in heifers, a lower (P less than .008) percentage composition of histological type I and II luteal cells in cows and a lower (P less than .0001) in vitro P4 response to LH in both cows and heifers when compared with the CL formed following spontaneous estrus. Cows had heavier (P less than .002) CL with a higher (P less than .05) number of large cells, a higher (P less than .09) percentage composition of histological cell type I and II luteal cells, and a higher (P less than .03) in vitro basal and stimulated P4 secretion. It is concluded that 1) the events associated with artificially shortening the estrous cycle with PGF altered subsequent CL function in Brahman females; 2) cows had heavier CL, which were composed of more steroidogenically functional luteal cells, than heifers and 3) age of animal interacted with PGF to alter subsequent CL function in Brahman females.     

Involvement of high-density lipoprotein in stimulatory effect of hormones supporting function of the bovine corpus luteum

The hypothesis that epinephrine (noradrenaline, NA) enhances utilisation of high-density lipoproteins (HDL) by bovine luteal cells and that this process involves phospholipase (PL) C and protein kinase (PK) C intracellular pathway was tested. Luteal cells from days 2-4, 5-10 or 11-17 of the oestrous cycle were preincubated for 20 h. Subsequently DMEM/Ham's F-12 medium was replaced by fresh medium and the cells were treated for 6 h as follows: In Experiment I with HDL (5-75 micrograms cholesterol per ml), NA, isoprenaline (ISO) or luteinising hormone (LH). In Experiment II cells were incubated for further 24 h in deficient medium (without FCS) and next treated as in Experiment I. In Experiment III cells were stimulated with NA, ISO or LH alone and together with HDL. In Experiment IV cells were treated with PLC inhibitor (U-73122) or with PKC inhibitor (staurosporine) or stimulator (phorbol 12-myristrate 13-acetate) and with either NA, insulin or LH. Only luteal cells from days 5-10 of the cycle responded on HDL and beta-mimetics (P < 0.05). LH stimulated progesterone secretion from the luteal cells during all stages of the cycle (P < 0.001). Cells incubated in deficient medium and supplemented with HDL secreted as much progesterone as those stimulated by LH in all stages of the cycle. Beta-mimetics were unable to enhance the stimulatory effect of HDL. Blockade of PLC had no influence on progesterone secretion from cells treated with either NA or LH, but this did impair the stimulatory effect of insulin (P < 0.05). Similarly, blockade of PKC by staurosporine impaired (P < 0.05) the effect of insulin only but not that observed after LH or NA treatment. We suggest that: (a) noradrenergic stimulation does not enhance utilisation of cholesterol from HDL for progesterone secretion; (b) the fasting of luteal cells seems to activate enzymes responsible for the progesterone synthesis; (c) effect of NA on progesterone secretion from luteal cells does not involve the PLC-PKC pathway.     

A review of lipoprotein cholesterol metabolism: importance to ovarian function

Cholesterol utilized for steroid synthesis by ovarian tissue may be derived from de novo synthesis or cellular uptake of lipoprotein cholesterol. The majority of blood cholesterol is transported by either low (LDL) or high (HDL) density lipoproteins, depending on the animal species. Prior to vascularization, only HDL are in follicular fluid and contribute sterol to granulosa cells because other lipoproteins are unable to transverse the basement membrane due to their molecular masses. Following vascularization, both LDL and HDL bathe luteal cells. Most species preferentially use LDL cholesterol as a precursor for ovarian steroid synthesis. The LDL uptake by ovarian tissue occurs by receptor-mediated endocytosis. The receptor recognizes apolipoprotein B of LDL and apolipoprotein E found on some, but not all, HDL. Within a species, a positive relationship may exist between HDL apolipoprotein E content and importance of HDL cholesterol as a precursor for steroidogenesis. A "HDL pathway" exists for uptake of sterol from HDL void of apolipoprotein E. The HDL receptor exhibits broad binding specificity. Unlike LDL, the HDL particle is not internalized, and cholesterol preferentially is taken up relative to other HDL constituents. In most species, lipoproteins, rather than de novo synthesis from acetate, contribute the majority of cholesterol used for steroid production. Trophic hormones increase lipoprotein binding, internalization, degradation and conversion of lipoprotein-derived sterol to steroids, effects that are mediate through cyclic adenosine monophosphate. Knowledge recently acquired regarding lipoprotein sterol utilization by the ovary may be useful in developing nutritional, pharmacological or endocrine manipulations that may positively affect cholesterol clearance by the ovary, steroidogenesis and reproductive performance.     

The effect of Eimeria acervulina infection on plasma lipids and lipoproteins in young broiler chicks

In young broiler chicks inoculated with 2 x 10(6) sporulated oocysts of Eimeria acervulina per bird, total plasma lipids were significantly depressed compared with controls in the first week after inoculation. The lowest level observed was at 5 days post-inoculation (d.p.i.), at which time the chick host is known to experience malabsorption in the chick host (Ruff and Wilkins, 1980). Analysis of plasma components of infected chicks at 4 and 7 d.p.i. showed that triglycerides, total cholesterol, free fatty acids, pigments and total protein were significantly decreased compared with controls. At 7 d.p.i., reduction of total cholesterol reflected mainly reduction in high density lipoprotein (HDL) cholesterol. However, the ratio of HDL cholesterol/total plasma cholesterol was not significantly different from the control ratio. Density gradient ultracentrifugation of chick plasma separated lipoproteins into three main fractions: portomicrons plus very low density lipoproteins (PM + VLDL), low density lipoproteins (LDL) and HDL. These fractions were analyzed for lipid content. Infection with E. acervulina caused (1) significant reduction in the triglyceride and cholesterol contents of the PM + VLDL fraction at 3 and 5 d.p.i., (2) significant reduction of LDL cholesterol at 9 d.p.i. and LDL phospholipid at 5-9 d.p.i., and (3) significant reduction of HDL cholesterol at 3-9 d.p.i. and HDL phospholipid at 5-9 d.p.i. Starvation of uninfected chicks for 48 h caused significant reduction in plasma triglycerides and phospholipids, but an increase in total cholesterol. Density gradient ultracentrifugation showed that the changes in these components reflected mainly reduction of the lipids in the PM + VLDL fraction. The LDL fractions, however, appeared more intense than those of the controls and contained more cholesterol and phospholipids. These results suggest that changes at 3 and 5 d.p.i. in the plasma lipoprotein pattern of chicks infected with E. acervulina most closely resemble changes seen in chicks starved for 48 h as far as PM + VLDL fraction is concerned. However, changes seen from 7 to 9 d.p.i. involve the LDL and HDL fractions and may reflect alterations in lipid and/or lipoprotein synthesis in the liver and intestine.     

Oxidized-low density lipoprotein inhibits cyclic AMP production by porcine luteal cells

Oxidized(OX)-low density lipoprotein (LDL) inhibits steroidogenesis by luteal cells (LC) from regressing porcine CL. The present study was designed to investigate the mechanism of inhibition by determining whether OX-LDL inhibits basal and agonist-stimulated cAMP production in regressing LC. Collagenase-dispersed porcine LC (n = 7 animals, estrous cycle Day 12-15) were cultured (2.5 x 10(5) cells/0.5 ml) in serum-free DMEM/Hams F-12 in duplicate wells at 37 degrees C. Approximately 18 hr after plating, media were replaced and LC were immediately treated with human LDL (0, 25, or 100 microg/ml) or OX-LDL (25 or 100 microg/ml). LC were incubated for 2 hr before addition of isobutylmethylxanthine (IBMX) to inhibit phosphodiesterase activity, immediately followed by hCG (100 ng/ml), cholera toxin (CT; 0.1 microM), forskolin (FS; 50 microM), or no further treatment (controls). LC were incubated for an additional 90 min. After removal of culture media, cells were extracted with 0.1 N HCl. Cell extracts were assayed for cAMP by enzyme immunoassay (EIA). HCG, CT, and FS increased (P < 0.05) cAMP production approximately four-, 10-, and 25-fold, respectively, relative to controls. OX-LDL (25 and 100 microg/ml) inhibited (P < 0.05) cAMP production by unstimulated, hCG-, and CT-stimulated LC, but not that by FS-stimulated LC. The highest concentration of OX-LDL (100 microg/ml) reduced cAMP formation by 39.8 +/- 6.6%, 44.7 +/- 10.5%, and 67.7 +/- 4.5% in unstimulated, hCG-, and CT-stimulated LC, respectively. In contrast, unmodified LDL (25 and 100 microg/ml) did not alter cAMP production. We conclude that OX-LDL can interfere with the cAMP signaling pathway in regressing luteal cells by acting at sites proximal to adenylate cyclase activation.     

The isolation, characterisation and quantification of the equine plasma lipoproteins

Plasma lipoproteins were isolated from eight Thoroughbred horses and eight Shetland ponies on the basis of particle size by gel filtration chromatography and according to density using rate-zonal ultracentrifugation. Three major classes corresponding to very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were identified and characterised by their lipid and apolipoprotein compositions. The particle size distributions of each class were determined by electron microscopy and non-denaturing polyacrylamide gradient gel electrophoresis. HDL was found to dominate the equine lipoprotein spectrum, accounting for 61 per cent of the total plasma lipoprotein mass (VLDL 24 per cent, LDL 15 per cent). The VLDL class was isolated as a single population of particles that were triglyceride rich and cholesterol, phospholipid and protein poor. Equine LDL was characteristically cholesterol rich and was found to be polydisperse comprising three subfractions that were discrete with respect to particle size and lipid composition. The HDL class was composed of homogeneous particles that were typically protein rich. Apolipoprotein (apo) B was the major protein of VLDL and LDL, and presented two components on polyacrylamide gel electrophoresis with molecular weights in the region of human apoB-100 and a third in VLDL similar to that of apoB-48. ApoA-I was the predominant protein in equine HDL. Although there were no breed differences in the physical or chemical properties of each lipoprotein class, the Shetland ponies had higher plasma triglyceride and VLDL concentrations than their Thoroughbred counterparts.     

Changes and interrelationships among luteal LH receptors, adenylate cyclase activity and phosphodiesterase activity during the bovine estrous cycle

Changes and interrelationships among the cellular mechanisms that may regulate luteal progesterone synthesis during the bovine estrous cycle were studied. Corpora lutea (CL) were enucleated from 30 cows via a transvaginal incision on d 4, 7, 10, 13, 16 and 19 following estrus (estrus = d 0). Mean corpus luteum weight, and luteal progesterone and plasma progesterone concentrations increased (P less than .05) from d 4 to 7 or 10, and declined following luteal regression (d 19). Unoccupied LH receptor (UOR) concentrations increased (P less than .05) more than fourfold from d 4 (38 fmol/mg protein) to d 16 (173 fmol/mg protein) before declining (P less than .05) by d 19 (30 fmol/mg protein). The dissociation constant for UOR concentrations increased (P less than .05) during the estrous cycle. Concentrations of occupied LH receptors (OR) were less (P less than .05) on d 7 than other days; however, the total number of OR/CL increased fourfold from d 4 (47 fmol/CL) to d 10 (221 fmol/CL) and remained similar thereafter. Basal adenylate cyclase, LH-activated adenylate cyclase, and Gpp(NH)p-activated adenylate cyclase activities were greatest (P less than .05) on d 7 to 16 as compared with d 4 and 19. Luteinizing hormone stimulated (P less than .05) adenylate cyclase activity relative to basal activity on d 7, 10, 13, and 16; whereas, Gpp(NH)p stimulated (P less than .05) adenylate cyclase activity relative to basal activity at each period. Phosphodiesterase was 46% greater on d 19 as compared with d 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of angiogenic activity and progesterone by ovine luteal cell types in vitro

In the first experiment, minced luteal tissues from cyclic ewes (n = 5) were incubated for 6 h. Media conditioned by these luteal tissue explants stimulated proliferation and migration of endothelial cells. In a second experiment, corpora lutea (CL) from superovulated ewes (n = 12) were dissociated (two ewes/dispersion) and separated into three fractions: a non-elutriated fraction containing a mixed population of luteal cells, a fraction enriched with small steroidogenic luteal cells, and a fraction containing primarily large steroidogenic luteal cells. Fractions (2 X 10(5) viable steroidogenic luteal cells per milliliter of medium) were incubated with LH in doses of 0, .1, 1, 10, and 100 ng/ml for 7 d. Conditioned media were collected on d 1, 3, 5, and 7 of incubation. Across all days of incubation, media from small luteal cells stimulated proliferation of endothelial cells. Media from large luteal cell incubations, however, secreted an endothelial mitogen only on d 7 of culture. Mixed luteal cell cultures secreted mitogenic activity on d 3, 5, and 7 of incubation, but not on d 1. Luteinizing hormone did not influence release of mitogenic activity by any luteal cell fraction. Across all days of incubation, media from large luteal cells contained more progesterone than those from small luteal cells (528 +/- 137 vs 48 +/- 16 ng/ml with no LH). Mixed (non-elutriated) and small luteal cells increased progesterone secretion in response to LH, and this response was maintained during long-term culture. Large luteal cells did not increase progesterone secretion in response to LH. Steroidogenic activity of all cell types decreased as incubation time progressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppressive action of gonadotropin-releasing hormone and luteinizing hormone on function of the developing ovine corpus luteum

Experiments were conducted to examine the effects of exogenous GnRH and LH on serum concentrations of progesterone (P4) in the ewe. Ewes in Exp. 1 and 2 were laparotomized on d 2 of an estrous cycle and ewes with corpora lutea (CL) in both ovaries were unilaterally ovariectomized. Ewes with CL in one ovary only were not ovariectomized. While they were anesthetized, ewes (n = 5) were injected with 25 micrograms GnRH (Exp. 1) or 50 ng GnRH (Exp. 2) into the artery supplying the ovary bearing the CL. Control ewes (n = 5 in each experiment) were injected similarly with saline. In Exp. 3, six ewes were injected i.v. (jugular) on d 2 with 100 micrograms oLH (t = 0) and 50 micrograms oLH at 15, 30 and 45 min; six control ewes were injected similarly with saline. Jugular blood was collected from all ewes at frequent intervals after treatment for LH analysis and on alternate days of the cycle through d 10 or 11 for P4 analysis. Treatment with 25 micrograms GnRH increased serum concentrations of LH at 15, 30, 45 and 60 min postinjection (P less than .001) and reduced serum concentrations of P4 on d 7 through 11 (treatment x day interaction; P less than .05). Injection with 50 ng GnRH caused a slight increase in serum concentrations of LH at 15 min but had no effect on serum concentrations of P4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Composition and electrophoretic mobility of plasma lipoproteins of dromedary camels (Camelus dromedarius)

OBJECTIVE: To determine the lipid composition and electrophoretic pattern of plasma lipoproteins in samples obtained from healthy 1-humped camels (Camelus dromedarius). ANIMALS: 34 healthy camels raised under similar farming and dietary conditions. PROCEDURES: Plasma samples were subjected to density-gradient ultracentrifugation for separation of plasma lipoproteins, including very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Purity of the separation was assessed by use of polyacrylamide gel disk electrophoresis. Concentrations of triglycerides, cholesterol, and phospholipids were measured in each lipoprotein fraction, and lipoprotein electrophoretic patterns were determined in plasma samples. RESULTS: Phospholipid was the major constituent of VLDL (mean +/- SD concentration, 10.62 +/- 1.2 mg/dL), LDL (24.66 +/- 3.12 mg/dL), and HDL (38.08 +/- 0.76 mg/dL). Low-density lipoprotein, VLDL, and HDL were important plasma lipoprotein carriers for cholesterol (67.94 +/- 9.51%), triglyceride (55.83 +/- 7.81%), and phospholipid (51.91 +/- 1.55%), respectively. On the basis of electrophoresis results, relative percentages of alpha- and beta-lipoproteins were 31.72 +/- 4.88% and 68.3 +/- 4.68%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The lipoprotein profile in 1-humped camels differed substantially from that of other ruminants. Results may be useful in the evaluation of metabolic disorders in camels.     

Tumor necrosis factor-α inhibits the stimulatory effect of luteinizing hormone and prostaglandin E(2) on progesterone secretion by the bovine corpus luteum

Tumor necrosis factor-α (TNF-α) is involved in the tissue remodeling that occurs in the corpus luteum (CL) during its development and regression. This cytokine is also implicated in the regulation of reproduction by its actions on ovarian steroidogenic cells. The aim of this study was to examine the influence of TNF-α on (1) progesterone (P(4)) output by the bovine CL and on (2) the responsiveness of the CL to LH or prostaglandin E(2) (PGE(2)) in vitro. In experiment 1, CL (days 8 to 10 of the estrous cycle) were perfused by using an in vitro microdialysis system with TNF-α (0.1, 0.5, or 1 μg/mL) alone or with TNF-α (1 μg/mL) followed by LH (1000 ng/mL) or PGE(2) (2 × 10(-5) M). Basal P(4) release (P < 0.05) was increased by TNF-α (0.5 or 1 μg/mL). Moreover, TNF-α (1 μg/mL) inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). In experiment 2, 4 h after intrauterine infusion of TNF-α (0.01 μg/mL or 1 μg/mL), CL (days 8 to 10 of the estrous cycle) were collected by colpotomy, cultured, and stimulated with LH (10 ng/mL) or PGE(2) (10(-6) M). Intrauterine infusion of TNF-α at a concentration of 1 μg/mL increased basal P(4) output by CL (P < 0.05). Moreover, the intrauterine infusion of TNF-α at a concentration of 0.01 μg/mL inhibited the stimulatory effect of LH or PGE(2) on P(4) output (P < 0.05). These results indicate that TNF-α (1) does not have an effect on the autonomous, pulsatile release of P(4); (2) increases P(4) secretion by bovine CL with increasing doses, and (3) reduces in a dose-dependent manner the responsiveness of CL to luteotropic factors both directly (after infusion to CL) and indirectly (after intrauterine infusion).     

Bovine lipoprotein and apolipoprotein profiles as influenced by sex and growth.

Three (intact) Angus males and females that were half-sibs and born within 21 d of each other were selected for this study. Each animal was bled periodically from birth to slaughter (18 mo) to determine the qualitative composition of plasma lipoproteins and apolipoproteins during its growth and development. Major components observed were: 1) very low-density (VLDL), 2) low-density (LDL), and 3) high-density lipoproteins (HDL). Individual amounts of triglycerides, cholesterol, and proteins for the VLDL were not different (P greater than .05) between sexes at any time during growth and development. At 1 yr of age and 15 mo of age, females had significantly larger (143.4 and 93.5 mg/dl) amounts of protein in the HDL than males (67.0 and 93.5 mg/dl), respectively. Within the male group, the LDL triglyceride concentration of calves was significantly (P less than .05) higher (7.4 mg/dl) than at all other bleeding times. Within the female group, cholesterol values for the VLDL were significantly (P less than .05) larger as calves and weanlings (16.5 and 21.7 mg/dl respectively) than for other bleeding periods. At all stages of growth and development, the HDL apoprotein profiles showed a distinct band with a weight of about 28,000 Da, which represented apolipoprotein-A-I. During the suckling stage, pooling of LDL fractions provided two components on the acrylamide gel (7.5 to 20%), apolipoprotein-B and a low molecular weight band. At 12 and 15 mo, no low molecular weight band was present in the pooled LDL fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipoprotein metabolism and hyperlipidaemia in the clog and cat: A review

The term hyperlipidaemia is used to describe raised plasma concentrations of cholesterol and, or, triglycerides. These aqueous insoluble lipids are transported through plasma in special particles called lipoproteins of which there are four main types; chylomicrons, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL). A transient rise in plasma triglycerides occurs following a meal as dietary fat is carried from the small intestine into the circulation by chylomicrons; this is called post prandial hyperlipidaemia. In addition, hyperlipidaemia is caused by defects in the metabolism of one or more of the lipoprotein classes which may be either genetic in origin or, more commonly in the dog and the cat, secondary to diabetes mellitus, hypothyroidism, hyperadrenocorticism, and renal and hepatic disease. Hypertriglyceridaemia is caused by reduced clearance of chylomicrons and VLDL, sometimes with overproduction of VLDL, whereas hypercholesterolaemia results from altered metabolism of LDL and HDL. Raised plasma triglycerides interfere with a number of clinical chemistry tests and may be associated with cutaneous xanthomata, vomiting and diarrhoea, peripheral nerve paralyses, seizures, pancreatitis, hepatosplenomegaly and lipaemia retinalis. The clinical manifestations of hypercholesterolaemia in the dog are few and largely confined to the eye. Diagnostic efforts should concentrate on determining whether the hyperlipidaemia is either genetic in origin or secondary to endocrine and systemic diseases. Plasma lipid concentrations usually return to normal with effective therapy of any underlying disease. Where no such disease can be identified, the hyperlipidaemia should be considered idiopathic in origin and the patient placed on a low fat diet.     

Quantitative analysis of canine plasma lipoproteins

A combined ultracentrifugationl/precipitation method for the measurement of lipoprotein cholesterol concentrations was developed and validated for use with canine plasma. Very low density lipoproteins (VLDL) were isolated by flotation ultracentrifugation and low density lipoproteins (LDL) separated from high density lipoproteins (HDL) by precipitation with heparin-manganese chloride. Effective separation of these classes was confirmed by agarose gel electrophoresis of native lipoproteins and by sodium dodecyl sulphate polyacrylamide gel electrophoresis of their apolipoprotein distributions. There was trace contamination of the LDL precipitate with HDL, but this represented less than 4 and 9 per cent of the total plasma HDL in normo- and hypercholesterolaemic dogs, respectively. The intra-assay and interassay coefficients of variation for LDL- and HDL-cholesterol concentrations were between 3·3 and 6·9 per cent, and 7·2 and 9·0 per cent, respectively, for plasma cholesterol concentrations between 2·67 and 8·14 mmoll/litre. The intra-assay coefficient of variation for VLDL-cholesterol was 53·8 and 18·4 per cent at plasma cholesterol concentrations of 2·67 and 8·14 mmol/litre, respectively. The interassay coefficient of variation for VLDL was 22·5 per cent. Storage of plasma at -20°C for between two and eight weeks did not affect VLDL-cholesterol concentrations, but led to an increase in LDL-cholesterol and a decrease in HDL-cholesterol concentrations of approximately 10 per cent. The method described is appropriate for the measurement of lipoprotein concentrations in plasma from normo- and hypercholesterolaemic dogs, but samples should not be subjected to prolonged storage before analysis.     

Influence of polychlorinated biphenyls on LH-stimulated secretion of progestereone and oxytocin from bovine luteal cells

Polychlorinated biphenyls (PCBs) due to their lipophilic properties can be easily accumulated in animal and human body and elicit diverse effects causing impairment of reproductive processes. Since these compounds were not be able to affect directly the luteal steroidogenesis, the aim of the present study was to verify hypothesis that PCBs can impair the effect of LH on the secretory function of luteal cells. Bovine luteal cells from different stages of the oestrous cycle (days 1-5, 6-10, 11-15 and 16-18) were exposed for 72h to various congeners of PCBs (PCB 126, PCB 77 and PCB 153) at the doses of 1, 10 or 100 ng/ml, in the presence or absence of LH (100 ng/ml), to determine the possible effect of these compounds on progesterone (P4) and ovarian oxytocin (OT) secretion. Only PCB 77 on days 1-5 and 16-18 increased P4 secretion. All PCBs decreased LH-simulated secretion of P4 from luteal cells obtained from all days of luteal phase. Dioxin-like congener (PCB 126) inhibited (P<0.05) the most evidently LH effect on P4 secretion. All congeners, except the lower doses of PCB 126, increased (P<0.05) OT secretion. They can also increase LH-stimulated secretion of OT, but the effect was dependent on the congener used and on the phase of oestrous cycle. On days 1-5 and 10-15, PCB 126 diminished LH-stimulated effect on OT secretion from luteal cells. PCB 77 (mimickig both dioxin and estradiol effect) in the higher doses, amplified effect of LH-stimulated OT secretion, while on all other days it diminished LH influence. PCB 153, which has estrogen-like properties, amplified LH effect on OT secretion during all studied days of the cycle. We conclude that PCBs (supposedly via estrogen and arylhydrocarbon - AhR receptor) may directly affect LH-stimulated function of CL. This does not appear to be a direct adverse effect on luteal steroidogenesis, but rather indirect on OT secretion from or within CL.     

Expression of proopiomelanocortin, proenkephalin and prodynorphin genes in porcine luteal cells

The objective of the study was to examine the expression of the genes coding for proopiomelanocortin (POMC), proenkephalin (PENK) and prodynorphin (PDYN) in porcine luteal cells isolated from corpora lutea (CL) collected on days 3-6, 8-10 and 13-16 of the oestrous cycle. Total RNA was purified from non-incubated cells and from cells incubated for 48 h in the absence or presence of luteinising hormone (LH). The semi-quantitative RT-PCR technique, involving coamplification of the target and control cDNA (beta-actin or 18S rRNA), was used to examine gene expression. It was found that the genes coding for opioid precursors are expressed in both non-incubated and incubated porcine luteal cells representing the early, mid- and late luteal phase. In non-incubated cells, only POMC mRNA content changed during CL development, whereas the expression of PENK and PDYN genes remained relatively constant. Additionally, the treatment of cells with LH markedly affected the expression of POMC and PENK, but no influence on PDYN expression was observed. The present study indicates that porcine luteal cells may produce opioid peptides and that gene expression of their precursors (except for PDYN) may be modulated in these cells by LH. Moreover, the present results support the involvement of opioid peptides in local regulation within the CL of the pig.     

Endocrine and cellular characteristics of corpora lutea from cows with a delayed post-ovulatory progesterone rise

The timing of the post-ovulatory progesterone rise is critical to the embryonic development and survival. The aim of this study was to determine the underlying causes of delayed post-ovulatory progesterone rises. Two groups of non-lactating dairy cows with early (n = 11) or late (n = 9) post-ovulatory progesterone rises were created by inducing luteolysis in the presence of either a large (> 10 mm) or small (< 10 mm) follicle, respectively. LH pulses were measured on days 4 (all cows) and 7 (n = 7, early; n = 5, late) (day 1= ovulation). The cows were slaughtered on day 5 (n = 4 each group) or 8 (n = 7, early; n = 5, late). Immunohistochemical analysis for endothelial cells (von Willebrand Factor, VWF), steroidogenic cells (3beta-HSD) and proliferation marker (Ki67) were performed. The basal progesterone production and LH responsiveness (0.001-100 ng/ml) of dispersed luteal cells was investigated. The luteal concentrations of FGF-2 and VEGF were measured by ELISA and RIA, respectively. There were no differences in LH pulse characteristics, area of VWF staining, proliferation index, steroidogenic cell characteristics, basal or LH-stimulated progesterone production by luteal cells between cows with an early or late progesterone rise (P > 0.10). However, the area of VWF staining increased from days 5 to 8, while the proliferation index decreased (P < 0.05). Furthermore, the luteal cells were more responsive to LH on day 8 (P < 0.01). Luteal concentrations of FGF-2 were higher on day 5 (P = 0.05), while VEGF was greater on day 8 (P < 0.01). In conclusion, we have clearly shown that LH support, degree of vascularization or luteal cell steroidogenic capacity were not the major factors responsible for inadequate secretion of progesterone by the developing bovine CL.     

Luteinizing hormone receptor number and affinity in corpora lutea from prepuberal gilts induced to ovulate and spontaneous corpora lutea of mature gilts

This study was designed to determine if luteal cell receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) contribute to the previously demonstrated abnormal function of induced corpora lutea (CL) in gilts. Twenty-five prepuberal (P) gilts, induced to ovulate with 1,500 IU pregnant mare serum gonadotropin followed 72 h later with 500 IU hCG (d 0 = day of hCG), and 22 mature (M) gilts that had displayed two or more estrous cycles were ovariectomized (OVX) on d 10, 14, 18, 22 or 26 after the onset of estrus. All gilts except those OVX on d 10 were hysterectomized between d 6 and 9 to ensure luteal maintenance. The CL were stored at -196 degrees C until determination of LH/hCG receptor number and dissociation constant (KD) by saturation analysis. Receptor number was greater for M than for P gilts on d 14 (P less than .07) and d 18 (P less than .01). The KD was greater in M than in P gilts on d 14 (P less than .01) and d 18 (P less than .0001). The LH/hCG receptor number and KD of P gilts remained the same throughout the days studied. The LH/hCG receptor number (fmol/mg protein) of M gilts was elevated on d 10, 14, and 18 (50.8, 50.4 and 51.4, respectively) and decreased on d 22 (26.5) and d 26 (25.4) to values similar to those of P gilts. In M gilts, KD increased on d 14, remained high on d 18 and decreased on d 22. We suggest that abnormal function of induced CL in P gilts may be due to an elevated LH receptor number.