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.     

Nitric oxide induces apoptosis in bovine luteal cells

We previously showed in in vivo and in vitro studies that nitric oxide (NO) is engaged in luteolysis in cattle. Nitric oxide produced locally in the bovine corpus luteum (CL) inhibits progesterone (P4) synthesis and is suggested to be a component of the luteolytic cascade induced by uterine prostaglandin (PG) F2alpha. In the present study, the molecular mechanisms of NO action during structural luteolysis were studied in cultured bovine luteal cells (Days 15-17 of the estrous cycle). The effects of the NO donor (NONOate; 10(-4)M) on DNA fragmentation, cell viability, P4 production and caspase-3 activity were compared with those of PGF2alpha (10(-6)M). Moreover, mobilization of intracellular calcium [Ca2+]i and gene expressions of Fas-L, Fas, bcl-2, bax, and caspase-3 in the cells were determined by semi-quantitative RT-PCR after NONOate treatment. Caspase-3 activity was examined calorimetrically. Contrary to PGF2alpha NONOate decreased cell viability. DNA fragmentation after NONOate treatment increased by more than with PGF22alpha. NONOate increased mobilization of [Ca2+]i in the cells. Although the NO donor did not affect Fas-L and bcl-2 gene expression, it stimulated Fas and bax mRNA and caspase-3 expression. The ratio of bcl-2 to bax mRNA level decreased in the cells treated with NONOate. Moreover, NONOate stimulated caspase-3 activity more effectively than PGF2alpha. The overall results suggest that NO is a luteolytic factor that plays a crucial role in regulation of the estrous cycle in structural luteolysis by inducing apoptosis of luteal cells in cattle.     

Effects of concanavalin A on the progesterone production by bovine steroidogenic luteal cells in vitro

The aim of this study was to evaluate the effects of concanavalin A (CONA) on the progesterone (P4) production by bovine steroidogenic luteal cells (LCs) in vitro. Luteal cells were collected during the mid‐luteal stage (at 10–12 days following ovulation) and processed in the laboratory. Luteal cells were grown for 7 days in a humid atmosphere with 5% CO₂, with or without 10% foetal bovine serum, and were subjected to the following treatments: control: no treatment; CONA (10 μg/ml); LH (100 μg/ml); CONA + LH; LH (100 μg/ml) + prostaglandin F2α (PGF2α) (10 ng/ml); CONA + LH + PGF2α. Samples of the culture media were collected on days 1 (D1) and 7 (D7) for P4 quantification. The cells were counted on D7 of culture. Differences between treatments were considered statistically significant at p < .05. Culture in the presence of CONA decreased the P4‐secreting capacity of LCs on D7 of culture, particularly in the absence of serum. The cell numbers did not change between treatments.     

Inhibition of bovine luteal function by indomethacin

Experiments were conducted to determine the effects of infusing indomethacin, a prostaglandin synthetase inhibitor, into the uterine lumen on the development and function of the bovine corpus luteum in the presence and absence of concurrently administered oxytocin. Each treatment was given twice daily on d 4, 5 and 6 of the estrous cycle. Treatments (six heifers/group) and resulting estrous cycle lengths were as follows: (1) untreated controls, 20.6 +/- .4 d; (2) .2 M phosphate buffer vehicle infused into the uterine lumen, 21.0 +/- .6 d; (3) 40 mg indomethacin infused into the body of the uterus, 16.5 +/- 1.0 d; (4) 150 USP units oxytocin injected sc, 10.0 +/- 1.2 d and (5) a combination of oxytocin and indomethacin as in treatments 3 and 4, 14.1 +/- 1.3 d. Plasma concentrations of progesterone were lower (P less than .05) in each treatment group from d 7 onward, when compared with untreated and vehicle-treated controls. Indomethacin alone effectively inhibited the development and function of the corpus luteum, and was without effect on oxytocin-induced inhibition of luteal function. In summary, it appears that a prostaglandin of either uterine or ovarian origin, or both, is required for the normal development and function of the bovine corpus luteum.     

Possible roles of intracellular cyclic AMP, protein kinase C and calcium ion in the apoptotic signaling pathway in bovine luteal cells

Structural luteolysis occurs by apoptosis of luteal cells. The present study examined the effects of activators of well-characterized second messengers on Fas and caspase-3 mRNA expression and on P4 production in luteal cells in order to trace the pro- and anti-apoptotic factors in the bovine corpus luteum (CL). Cultured bovine mid luteal cells were treated for 24 h with a cyclic AMP analogue (8-bromo cyclic AMP; 8br-cAMP; 2.5 mM), a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate; PMA; 10 microM), or calcium ionophore (A23187; 10 microM). Fas and caspase-3 mRNA expression was inhibited by 8br-cAMP and PMA but was increased by A23187 (P<0.05). In addition, P4 production by bovine luteal cells was stimulated by 8br-cAMP and PMA, whereas it was inhibited by A23187, compared with untreated controls (P<0.05). The overall results suggest that cAMP and PKC suppress apoptosis in bovine luteal cells through inhibition of Fas and caspase-3 mRNA expression and through stimulation of P4 production. Therefore, substances that activate cAMP or PKC may act as survival factors in the bovine CL. Furthermore, substances that mobilize Ca2+ may act as apoptotic factors by stimulating Fas and caspase-3 expression in the bovine luteal cells.     

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)     

Failure of neuropeptide y to modulate the release of LH and prolactin by cultured bovine pituitary cells

Possible direct effects of neuropeptide Y (NPY) on dispersed and cultured cells of the anterior lobe (AL) of the bovine pituitary were investigated. AL tissue from steers was enzymatically dissociated into individual cells, preincubated for 18 hr and then incubated in suspension cultures for 2 hr or 24 hr with either NPY, gonadotropin-releasing hormone (GnRH) or both. Release of luteinizing hormone (LH) and prolactin (PRL) into medium was quantified by radioimmunoassay and expressed as hormone released per 100,000 cells. Basal release of LH averaged 38 and 86 ng for 2 hr and 24 hr respectively while that of PRL averaged 118 and 438 ng for the same incubation periods. Addition of NPY did not alter (P>.05) basal release of LH or PRL for either duration of incubation. Also, NPY did not affect (P>.05) release of LH in response to GnRH. In summary, this study indicated that NPY, at in vitro dosages of .01 to 100nM, does not modulate the release of LH or PRL at the pituitary level in castrate cattle.     

The luteotrophic function of galectin-1 by binding to the glycans on vascular endothelial growth factor receptor-2 in bovine luteal cells

The corpus luteum (CL) is a temporary endocrine gland producing a large amount of progesterone, which is essential for the establishment and maintenance of pregnancy. Galectin-1 is a β-galactose-binding protein that can modify functions of membrane glycoproteins and is expressed in the CL of mice and women. However, the physiological role of galectin-1 in the CL is unclear. In the present study, we investigated the expression and localization of galectin-1 in the bovine CL and the effect of galectin-1 on cultured luteal steroidogenic cells (LSCs) with special reference to its binding to the glycans on vascular endothelial growth factor receptor-2 (VEGFR-2). Galectin-1 protein was highly expressed at the mid and late luteal stages in the membrane fraction of bovine CL tissue and was localized to the surface of LSCs in a carbohydrate-dependent manner. Galectin-1 increased the viability in cultured LSCs. However, the viability of LSCs was decreased by addition of β-lactose, a competitive carbohydrate inhibitor of galectin-1 binding activity. VEGFR-2 protein, like galectin-1, is also highly expressed in the mid CL, and it was modified by multi-antennary glycans, which can be recognized by galectin-1. An overlay assay using biotinylated galectin-1 revealed that galectin-1 directly binds to asparagine-linked glycans (N-glycans) on VEGFR-2. Enhancement of LSC viability by galectin-1 was suppressed by a selective inhibitor of VEGFR-2. The overall findings suggest that galectin-1 plays a role as a survival factor in the bovine CL, possibly by binding to N-glycans on VEGFR-2.     

Relationships between LH and estradiol-17 beta after removal of luteal progesterone in the ewe

Three experiments were conducted to examine the relationship between systemic concentrations of luteinizing hormone (LH) and estradiol-17 beta (E2) after withdrawal of progesterone in cycling ewes. In Exp. 1, ewes were assigned randomly to one of three treatments: laparotomy (C), removal of the luteal ovary (ULO), or ULO plus anesthesia with sodium pentobarbital for 6 h beginning 4 h after surgery. Anesthesia was used in an attempt to block the expected increase in tonic secretion of LH. Patterns of LH and E2 in these three groups did not differ during the 24-h experimental period. In Exp. 2, a longer period of anesthesia was utilized. Forty-eight ewes were assigned at random to one of four treatments: C, ULO, lutectomy or an intrafollicular injection of prostaglandin F2 alpha (PGF2 alpha). One-half of the ewes in each group were anesthetized with sodium pentobarbital from initiation of treatment (0 h) until 10 h after surgery. Sodium pentobarbital did not suppress the increases in LH and E2 after progesterone withdrawal. The regression of concentrations of E2 on concentration of LH was not significant. In Exp. 3, ewes were infused with either saline or dopamine after receiving an im injection of PGF2 alpha. Tonic secretion of LH increased after 4 h in ewes infused with saline, but not in ewes infused with dopamine. Despite the suppression of LH, concentrations of E2 increased in dopamine-treated ewes as in control ewes. Therefore, the initial increase in E2 after a decline of progesterone in cycling ewes is independent of increases in LH.     

The effects of luteinizing hormone as a suppression factor for apoptosis in bovine luteal cells in vitro

The fate of the corpus luteum, a transient endocrine gland formed and degraded during an oestrous cycle, is decided by various physiological factors, such as luteinizing hormone (LH). As a stimulator of progesterone, LH is known to maintain corpus luteum functional and structural integrity by inhibiting apoptosis, a programmed cell death. Therefore, we aim to investigate its action during the mid-luteal phase hypothesized that LH suppresses the death mechanism of bovine luteal steroidogenic cells (LSC) by analysing the expression of proteins involved. Cultured bovine LSC obtained from corpus luteum were treated for 24 hr with recombinant TNF and IFNG in the presence or absence of LH. The result showed that LH proved to have a protective effect by increased cell viability (p < .05) and prevented DNA fragmentation (p < .05), as demonstrated by the WST-1 colorimetric assay and TUNEL assay. Expression analysis of mRNA and protein levels showed that LH altered the expression of BCL2 (p < .05), CASP3 (p < .05), FAS (p < .05), and BAX (p < .05) to support cell survival. In conclusion, our study suggests that LH prolongs the corpus luteum life span through the anti-apoptotic mechanism by increasing cell viability and suppressing apoptosis-related genes and protein expression.     

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.     

Escherichia coli isolated from bovine mastitis invade mammary cells by a modified endocytic pathway

Escherichia coli is a major pathogen in the aetiology of bovine mastitis. Although classically considered to be an environmental pathogen causing mainly transient infection, the incidence of persistent E. coli mastitis infections may be increasing, suggesting an adaptation of this pathogen to the bovine udder environment. Mastitis E. coli strains have been demonstrated to enter bovine mammary cells in vitro but little is known about the invasion mechanism or the intracellular fate of the bacteria. In order to further understand the pathogenesis of persistent E. coli bovine mastitis we investigated the intracellular trafficking of mastitis E. coli isolates in primary bovine mammary cells using confocal microscopy and fluorescent markers of endocytic compartments. Consistent with other studies, mastitis E. coli were found to invade primary bovine mammary cells in vitro. This process did not involve in the rearrangement of the actin cytoskeleton. Intracellular bacteria were observed within membrane-bound compartments that labelled with the early endosomal marker phosphatidylinositol 3-phosphate (PtdIns(3)P) and also within late endosome-like compartments labelled with the small GTPase Rab7, indicating an endocytic mechanism of bacterial internalization. Bacteria were not observed within acidified lysosomal compartments or autophagic vacuoles, suggesting that the internalized bacteria are not targeted for lysosomal degradation via either the classical endocytic pathway or the autophagic response. Our findings are consistent with an endosomal survival niche for the internalized bacteria, allowing them to evade host immune responses and establish an infection reservoir that could later re-emerge as a recurrent clinical mastitis episode.     

The stimulation of testosterone and LH secretion by synthetic GnRH in the male Cape porcupine

The effects of GnRH stimulation on plasma testosterone and luteinizing hormone (LH) levels in Cape porcupine males were examined by analysing plasma collected before and after an intravenous injection of GnRH. In six mature males and one subadult, which were given an intravenous injection of 0,5 ml saline, levels of plasma testosterone and LH did not increase. Four weeks later an intravenous GnRH challenge (40 μ?) caused plasma testosterone to rise three-fold and LH to rise 10-15-fold within 180 min in five of the mature males. Peaks of plasma testosterone and LH occurred 90 and 120 min, respectively, after stimulation, and baseline and peak levels of both hormones were significantly related.     

Stimulation of bovine luteal oxytocin secretion in vitro by a phorbol ester and calcium ionophore

Experiments were conducted to examine the in vitro effects of a phorbol ester and a calcium ionophore on bovine luteal oxytocin (OT) secretion and synthesis and progesterone secretion. Corpora lutea removed from beef heifers on d 8 of an estrous cycle were sliced and incubated for 2 h with .81 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), 1.62 nM TPA or .3 microM calcium ionophore A23187. Both concentrations of TPA increased (P less than .01) OT secretion (ng.g-1.2 h-1; control, 407.1; .81 nM TPA, 494.7; 1.62 nM TPA, 528.1; SE = 21.2). Increased secretion of OT was accompanied by a corresponding increase (P less than .02) in synthesis of the hormone (ng.g-1.2 h-1; control, 368.5; .81 nM TPA, 427.6; 1.62 nM TPA, 492.1; SE = 25.7). Phorbol ester also induced (P less than .025) progesterone secretion (ng.g-1.2 h-1; control, 1,056.2 vs .81 nM TPA, 1,333.3; SE = 86.4). Calcium ionophore increased (P less than .01) OT secretion (ng.g-1.2 h-1; control, 248.9 vs A23187, 327.4; SE = 16) and there was a trend (P = .09) toward increased synthesis of OT in response to the ionophore (control, 124.4 vs A23187, 165.6; SE = 16.4). Because TPA can activate protein kinase C and A23187 increases intracellular calcium, these intracellular constituents probably are involved in promoting secretion of OT and progesterone.     

LH release from dispersed bovine pituitary cells in culture: in vitro effects of estradiol and procedural variables

Procedures for cell dissociation and in vitro culture were validated to investigate secretion of luteinizing hormone (LH) from bovine anterior lobe (AL) pituitary cells. The concentration of trypsin used for dissociation affected cell yield, cell loss during preincubation, LH secretion, and response to gonadotropin-releasing hormone (GnRH). Optimum results were obtained with trypsin concentrations of 8-16 micrograms/mg fresh tissue. Duration of preincubation and of experimental culture markedly affected LH secretion and response to GnRH. Immediately after dissociation, cells contained relatively low quantities of LH, but they were able to release a substantial proportion of this LH. Basal release of LH and GnRH-induced release of LH were highly correlated with total quantities of LH, and all three parameters increased with time of preincubation until 24 hr. Experimental treatments of 2 hr duration were optimal for investigating GnRH stimulation of LH release, whereas longer treatments may be required to investigate effects of agents that inhibit the release of LH. Preincubation of dissociated AL cells with physiological concentrations of estradiol increased all three LH parameters. Progesterone had no effect either alone or in combination with estradiol. In conclusion, the procedures described for cell dissociation and culture of suspended cells provide a useful tool for studying release of LH from the bovine AL cell.     

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.     

Influence of polychlorinated biphenyls on the secretion of oxytocin from bovine luteal cells and from granulosa cells obtained from the follicles of different size

Effect of polychlorinated biphenyles (PCBs) on viability and secretory function of luteal and granulosa cells from mature cows was studied. Luteal cells from corpora lutea of different developmental stages and granulosa cells from follicles of >1 cm< in diameter were used. Neither individual congeners (PCB-126, -77, -153) nor mixture of PCBs Aroclor Ar) 1248 at the dose of 1, 10 or 100 ng/ml affected the viability of cells (P>0.05) compared to control after 72 h of incubation. PCBs markedly increased (P<0.05-0.001) oxytocin (OT) secretion from granulosa cells. This effect was the most evident when granulosa cells from follicles <1 cm diameter was treated with PCB-77 which is assumed to stimulate both arylhydrocarbon receptor (AhR) and estradiol (E2) receptor. Even the lowest dose of this compound (1 ng/ml) outranged the effect produced by cortisol (10(-5)M) used as positive control. There was marked effect (P<0.05-0.001) of PCBs on luteal cells from days 6-15 of the estrous cycle. However, influence of PCBs on OT secretion from luteal cells on day 1-5 and 16-18 of the estrous cycle was less evident. Again, PCB-77 was the most efficient stimulator of OT secretion. While the lowest effect was found after treatment of cells with PCB-126 which has dioxin-like properties. It can be assumed that diverse effect of PCBs on female reproduction largely results from the influence of these compounds on ovarian OT secretion. Since both synthesis and secretion of ovarian OT in bovine do not markedly depend on estradiol, some alternative cellular pathways of PCBs on ovary function are suggested.