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1.
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.  相似文献   

2.
Prepuberal (P) gilts were induced to ovulate with pregnant mare serum gonadotropin followed 72 h later by human chorionic gonadotropin (hCG). Three P gilts and three mature (M) gilts each were ovariectomized on d 10, 14, 18, 22 and 26 (d 0 = day of hCG for P gilts and onset of estrus for M gilts). Gilts ovariectomized on d 14, 18, 22 and 26 were hysterectomized on d 6 to ensure maintenance of the corpora lutea (CL). Two to five grams of minced luteal tissue were dispersed using collagenase and hyaluronidase in HEPES buffered salt solution supplemented with glucose and bovine serum albumin. Dispersed cells were rinsed in Dulbecco's Modified Eagle Medium (DMEM), counted (ratio of large to total number of luteal cells determined) and then incubated for 1 h in DMEM. With aliquots standardized to 2.5 X 10(4) viable, large cells (greater than 25 micron diameter) were incubated in 1 ml DMEM for 2 h in the presence of either 10, 50, 100 or 1,000 ng luteinizing hormone (LH); .1, 1, 10 or 100 ng hCG; 10, 100 or 1,000 ng norepinephrine (NE) or either .75, or 1.5 mM dibutyrl cyclic adenosine monophosphate (dbcAMP). Progesterone (P4) in the medium was quantified by radioimmunoassay. Basal P4 production (no P4 stimulator added to the medium) on d 10, 14, 18, 22 and 26 for P gilts was 246 +/- 9, 66 +/- 4, 64 +/- 6, 41 +/- 3 and 69 +/- 6 ng/ml medium, respectively, and for M gilts was 281 +/- 12, 128 +/- 8, 53 +/- 4, 82 +/- 6, 101 +/- 5 ng/ml medium, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
The contractile effects of PGF2alpha (3 x 10(-6) to 10(-4) M) and PGE2 (10(-7) to 10(-5) M) were examined on isolated branches of ovarian artery (OA) and extramyometrial branches of uterine artery (UA) collected from pigs in the luteal (day 10-12) and follicular phase (day 17-20) of the estrous cycle, and during early pregnancy (day 10-12). Strong contraction was demonstrated in both arteries during all investigated periods in response to PGF2alpha, which was significantly higher (P < 0.01) than to PGE2, being negligible in the follicular phase. In UA, the effective dose of PGF2alpha (ED50) amounted 7.9 x 10(-6) M and 6.3 x 10(-6) M in the luteal and follicular phase, and 5.0 x 10(-6) M in early pregnancy. ED50 for PGE2 reached 5.0 x 10(-7) M in the luteal phase, and 4.1 x 10(-7) M in early pregnancy. For both prostaglandins, the contraction was much stronger (P < 0.01) in OA than in UA branches. In OA, the ED50 for PGF2alpha was 1.2 x 10(-5) M in the luteal phase and was significantly higher (P < 0.05) than in the follicular phase (3.1 x 10(-6) M) and early pregnancy (2.7 x 10(-6) M). ED50 for PGE2 amounted 7.3 x 10(-7) M in the luteal phase and 1.7 x 10(-7) M in early pregnancy. Studies showed the influence of the estrous cycle and early pregnancy on OA branches sensitivity to the contractile effect of PGF2alpha and the lack of this effect on UA branches, and the influence of the estrous cycle on UA and OA branch contraction in response to PGE2.  相似文献   

4.
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.  相似文献   

5.
The present studies were conducted: (1) to determine which beta-adrenoceptor subtypes are involved in progesterone and oxytocin (OT) secretion, (2) to examine whether noradrenaline (NA) acts directly on the cytochrome P-450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and (3) to study the effect of prostaglandin F2 alpha (PGF2 alpha) on NA-stimulated steroidogenesis in luteal cells. The effect of NA on progesterone secretion from luteal slices of heifers on days 8-12 of the oestrous cycle was blocked by both atenolol (beta 1-antagonist) and ICI 118.551 hydrochloride (beta 2-antagonist). OT secretion was blocked only after treatment with ICI 118.551 hydrochloride (P < 0.05). Dobutamine (10(-4)-10(-6) M), a selective beta 1 agonist and salbutamol (10(-4)-10(-6) M), a selective beta 2 agonist, both increased progesterone production (P < 0.01) with an efficiency comparable to that produced by NA (P < 0.01). The increase of OT content in luteal slices was observed only after treatment with salbutamol at the dose of 10(-5) M (P < 0.01). Dobutamine had no effect on OT production at any dose. A stimulatory effect of NA on cytochrome P-450scc activity (P < 0.05) was demonstrated using 25-hydroxycholesterol as substrate. 3 beta-HSD activity also increased following NA (P < 0.01) or pregnenolone (P < 0.05) and in tissue treated with pregnenolone together with NA (P < 0.01). PGF decreased progesterone synthesis (P < 0.05) and 3 beta-HSD activity (P < 0.01) in tissue treated with NA. We conclude that NA stimulates progesterone secretion by luteal beta 1- and beta 2-adrenoceptors, while OT secretion is probably mediated only via the beta 2-receptor. NA also increases cytochrome P-450scc and 3 beta-HSD activity. PGF inhibits the luteotropic effect of NA on the luteal tissue.  相似文献   

6.
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)  相似文献   

7.
Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.  相似文献   

8.
Nitric oxide induces apoptosis in bovine luteal cells   总被引：1，自引：0，他引：1  
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.  相似文献   

9.
Static primary cultures of bovine anterior pituitary (AP) cells were utilized to study the effect of sex steroids on basal growth hormone (GH) and GH-releasing hormone (GRF)-stimulated release of GH. The AP cells (5 x 10(5) cells/well) were allowed to attach for 72 hr and become confluent before treatments were imposed. Cells were incubated for an additional 24, 48 or 72 hr with either estradiol-17 beta (E2, 10(-11) to 10(-8) M), testosterone (T, 10(-8) to 10(-5) M), dihydrotestosterone (DHT, 10(-9) to 10(-6) M) or 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol, 10(-11) to 10(-8) M). Media were collected every 24 hr and GH concentrations determined by RIA. Incubation of calf AP cells with gonadal steroids did not affect (P > 0.05) basal GH released at 24, 48, or 72 hr. In another experiment, calf AP cells were incubated with the same concentrations of the steroids for 24 hr, media harvested, cells washed and challenged in serum-free media for 1 hr with bovine GRF 1-44-NH2 (10(-8) M). In non-steroid treated wells, GRF increased (P < 0.05) GH from 58 to 134 ng/ml. Incubation with E2 or 3 alpha-diol did not affect (P > 0.05) GRF-induced GH release; however, preincubation with T (10(-5) M) and DHT (10(-9), 10(-8) and 10(-7) M) increased (P < 0.05) GRF-induced GH release above control concentrations (195, 235, 190 and 185 ng/ml, respectively). At the doses tested, sex steroids did not affect basal release of GH, but androgens increased responsiveness of somatotropes to GRF.  相似文献   

10.
We have studied in the porcine endometrium the expression of oxytocin receptor (OTR) mRNA and the effect of progesterone (P4) on oxytocin/oxytocin receptor (OT/OTR) function concerning intracellular Ca2+ mobilisation ([Ca2+]i), prostaglandin F2alpha (PGF2alpha) and E2 (PGE2; PG) secretion. Tissue was taken from cyclic and early pregnant pigs (days 14-16). A higher expression of OTR mRNA (P < 0.05) was observed in the endometrium of cyclic than pregnant pigs. The stimulatory (P < 0.05) effect of OT (10(-7) M) on [Ca2+]i mobilisation was noticed within 15-60 s and 30-60 s in endometrial stromal cells of cyclic and pregnant pigs, respectively. In the presence of P4 (10(-5) M) basal and OT-stimulated [Ca2+]i concentrations decreased in stromal cells during luteolysis and pregnancy. In stromal cells P4 delayed mobilisation of [Ca2+]i in response to OT by 15 s during luteolysis and had no effect during pregnancy. In cyclic and pregnant epithelial cells OT stimulated mobilisation of [Ca2+]i in 45 s and 60 s, respectively. Oxytocin increased (P < 0.05) PGF2alpha secretion during luteolysis and pregnancy and PGE2 during luteolysis from endometrial slices. Progesterone did not inhibit this stimulatory effect. During luteolysis OT increased (P < 0.05) PGF2alpha in epithelial and stromal cells and PGE2 secretion in epithelial cells. In the presence of P4 this effect of OT was reduced only in stromal cyclic cells (6 h culture). The presence of P4 decreased the effect of OT on [Ca2+]i mobilisation only in stromal cells. We found that, in most conditions, P4 did not inhibit the OT-stimulated secretion of PG in the porcine endometrium.  相似文献   

11.
In the following investigations, the LH secretion of cells from pituitaries in heifers on days 16-18 of their oestrous cycle (n = 14) was analysed. Cells were dissociated with trypsin and collagenase and maintained in a static culture system. For the estimation of LH release, the cells were incubated with various concentrations of mammalian GnRH (Lutrelef) for 6 h. To determine the action of Antarelix (GnRH antagonist), the cells were preincubated for 1 h with concentrations of 10(-5) or 10(-4) M Antarelix followed by 10(-6) M GnRH coincubation for a further 6 h. At the end of each incubation, the medium was collected for LH analysis. Parallel, intracellular LH was qualitatively detected by immunocytochemistry. Changes in the intensity of LH staining within the cells in dependence of different GnRH concentrations were not observed, but a significant increase LH secretion in pituitary cells was measured at 10(-6) M GnRH. Antarelix had no effect on basal LH secretion at concentrations of 10(-4) and 10(-5) M. After coincubation of pituitary cells with Antarelix and GnRH, Antarelix blocked the GnRH-stimulated LH secretion with a maximal effect of 10(-4) M, but the staining of immunoreactive intracellular LH was detected at approximately the same level compared to the pituitary cells treated with exogenous GnRH alone. These data demonstrate that Antarelix is effective in influencing the GnRH-stimulated LH secretion of pituitary cells in vitro. After administration of Antarelix in vivo, the GnRH-stimulated LH secretion of cultured pituitary cells was not inhibited.  相似文献   

12.
We examined the effect of glucocorticoids on brush border membrane transporters and, furthermore, the involvement of Ca2+ in its action in the primary cultured rabbit renal proximal tubule cells (PTCs). Dexamethasone (DEX, 10(-9) M) decreased Pi uptake by 17%; whereas DEX affected neither alpha-methyl-glucopyranoside (alpha-MG) uptake nor Na+ uptake. The DEX-induced inhibition of Pi uptake was due to a decrease of V(max). In contrast, other steroid hormones such as progesterone, testosterone, and 17beta-estradiol (10(-9) M) did not induce inhibition of Pi uptake. In order to examine the involvement of Ca2+ in DEX-induced inhibition of Pi uptake, PTCs were treated with A 23187 (10(-6) M, Ca2+ ionophore). A 23187 also inhibited Pi uptake, mimicking DEX action in Pi uptake. Treatments with W-7 (10(-4) M, calmodulin dependent kinase inhibitor), KN-62 (10(-6) M, Ca2+/calmodulin-dependent protein kinase II inhibitor), and BAPTA/AM (10(-6) M) or TMB-8 (10(-4) M) (intracellular Ca2+ mobilization blockers) blocked the DEX-induced inhibition of Pi uptake. However, nifedifine, methoxyverapamil (10(-6) M, L-type Ca2+ channel blockers), and EGTA (1 mM, extracellular Ca2+ chelator) did not block it. In conclusion, DEX inhibited Pi uptake via, in part, Ca2+/calmodulin pathway mediated by intracellular Ca2+ mobilization in the PTCs.  相似文献   

13.
To evaluate the response of luteal cells to in vitro stimulation with luteinizing hormone (LH) or dibutyryl cyclic adenosine monophosphate (dbcAMP) and determine the secretion of progesterone by the fetoplacental unit, the corpora lutea were removed surgically in 10 cows (luteectomy) at 250 days (5 cows) or 270 days (5 cows) of gestation. During surgery, but before luteectomy, catheters were placed in the middle uterine artery and vein, carotid artery, and jugular vein. Blood samples were collected from all catheters just before luteectomy and at 8-hour intervals after luteectomy for 72 hours or until calving, whichever occurred first. Luteal tissue was prepared as a dispersed cell preparation and incubated with 0, 0.1, 1.0, 10, or 100 ng of LH/ml of medium or was incubated with 0, 0.5, or 2 mM dbcAMP. Synthesis of progesterone in response to LH by dispersed cells prepared from corpora lutea at 270 days was less (P less than 0.01; analysis of variance) than that by similar preparations at 250 days because a dose-response relationship was not observed for incubations of luteal tissue with LH at 270 days of gestation. Progesterone synthesis in response to the addition of dbcAMP also was less (P less than 0.01) at 270 than at 250 days of gestation. This difference in responsiveness to LH and dbcAMP between the 2 stages of gestation was not reflected by a significant difference between stages of gestation in systemic concentrations of progesterone before luteectomy.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
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.  相似文献   

15.
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.  相似文献   

16.
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.  相似文献   

17.
These experiments were undertaken to determine the potential for estradiol-17 beta (E2), 2-hydroxyestradiol-17 beta (2-OH-E2) and 4-hydroxyestradiol-17 beta (4-OH-E2) to regulate prostaglandin (PG) E and F2 alpha synthesis by pig endometrium. Endometrium was collected from pigs on d 10 of pregnancy and incubated (15 to 20 mg/well) for three 2-h periods in 2 ml of medium in 24-well culture plates. At the end of each period, the medium was removed and frozen. Later media were thawed and assayed for PGE and PGF2 alpha. During Periods 2 and 3, the medium contained 0, 25, 50, 100 or 150 microM 2-OH-E2 (Exp. 1); 0, 25 or 50 microM 4-OH-E2 (Exp. 2); or 0, 25 or 50 microM E2 (Exp. 3). Each experiment was a factorial with 2-OH-E2, 4-OH-E2 or E2 as one main effect and 0 or 1 mM ascorbate as a second main effect. Ascorbate decreased (P less than .01) PGE and PGF2 alpha release in all experiments. Two-hydroxyestradiol-17 beta decreased (P less than .01) PGE and PGF2 alpha release into the medium during Periods 2 and 3 in a dose-dependent manner (Exp. 1). In Exp. 2, 4-OH-E2 decreased (P less than .07) endometrial release of PGE and PGF2 alpha in Periods 2 and 3 and increased (P less than .01) the PGE:PGF2 alpha in Period 3. In Exp. 3, E2 decreased release of PGE during Period 3 and PGF2 alpha release during Period 2. The PGE:PGF2 alpha was not altered by E2.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

18.
In cattle, sub-luteal circulating progesterone induces an increase in the frequency of LH pulses, prolonged growth of the dominant follicle, increased peripheral estradiol and reduced fertility. The objective of this study was to examine the earliest stages of development of prolonged dominant follicles, to gain insight into the etiology of this aberrant condition. Heifers were treated with an intravaginal progesterone-releasing device (CIDR) from Day 4-8 post-estrus and PGF2alpha was injected on Day 6 and again 12h later (early prolonged dominant group). Follicular phase (CIDR: Day 4-6, with PGF2alpha) and luteal phase (CIDR: Day 4-8, without PGF2alpha) groups served as controls. As expected, peripheral progesterone in heifers of the early prolonged dominant group was intermediate between luteal and follicular phase groups after luteal regression (P<0.05). On Day 7, the frequency of LH pulses was higher in heifers of the follicular phase and early prolonged dominant groups than the luteal phase group (P<0.05). Dominant follicles (n = 4 per group) were collected by ovariectomy on Day 8 and were similar in size among groups (P>0.05). Estradiol and androstenedione concentrations in the follicular fluid at ovariectomy were higher in the follicular phase and early prolonged dominant groups versus the luteal phase group (P<0.01), whereas progesterone did not differ among groups (P>0.05). Granulosa cells and theca interna isolated from dominant follicles were incubated for 3h with or without gonadotropins or frozen for later analysis of mRNA for steroidogenic enzymes. Luteinizing doses (128 ng/ml) of LH and FSH increased secretion of progesterone (P<0.05) but did not affect secretion of estradiol by granulosa cells in all groups. Low (2 or 4 ng/ml) and luteinizing doses of LH increased secretion of androstenedione by theca interna to a similar extent among groups. Expression of mRNA for P450 side chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450 aromatase (aromatase) and Steroidogenic Acute Regulatory (StAR) protein by granulosa cells did not differ among groups (P>0.05). Levels of mRNA for P450scc, 3beta-HSD, 17alpha-hydroxylase (17alpha-OH) and StAR protein in theca interna were similar in the follicular phase and early prolonged dominant groups (P>0.05), but lower in the luteal phase group (P<0.05-0.1). In summary, the premature follicular luteinization observed in previous studies after prolonged periods of sub-luteal progesterone was absent in early prolonged dominant follicles, exposed to sub-luteal progesterone for 36 h, and their characteristics resembled those of control follicles during the follicular phase.  相似文献   

19.
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).  相似文献   

20.
Pituitary cells, from seven 160- to 170-day-old pigs, were studied in primary culture to determine the affects NPY on LH and GH secretion at the level of the pituitary. On day 4 of culture, medium was discarded, plates were rinsed twice with serum-free medium and cells were cultured in 1 ml fresh medium without serum and challenged individually with 10(-10), 10(-8) or 10(-6) M [Ala(15)]-h growth hormone-releasing factor-(1-29)NH(2) (GRF); 10(-9), 10(-8) or 10(-7) M GnRH or 10(-9), 10(-8), 10(-7) or 10(-6) M NPY individually or in combinations with 10(-9) or 10(-8) M GnRH or 10(-8) or 10(-6)M GRF. Cells were exposed to treatment for 4 h at which time medium was harvested and quantified for LH and GH. Basal LH secretion (control; n = 7 pituitaries) was 12 +/- 6 ng/well. Relative to control at 4 h, 10(-9), 10(-8) and 10(-7) M GnRH increased (P < 0.01) LH secretion by 169, 176 and 197%, respectively. Neuropeptide-Y did not alter (P > 0.4) basal LH secretion nor 10(-8) M GnRH-induced increase in LH secretion but 10(-9) M GnRH-stimulated LH secretion was reduced by NPY and was not different from control or GnRH alone. Basal GH secretion (control; n = 7 pituitaries) was 56 +/- 12 ng/well. Relative to control at 4 h, 10(-10), 10(-8) and 10(-6) M GRF increased GH secretion by 111%, 125% (P < 0.01) and 150% (P < 0.01), respectively. Only 10(-6) M (134%) and 10(-7) M (125%) NPY increased (P < 0.04) basal GH secretion. Addition of 10(-9), 10(-8) and 10(-7) M NPY in combination with 10(-8) M GRF suppressed (P < 0.04) GRF-stimulated GH secretion. However, 10(-9) M NPY enhanced (P < 0.06) the GH response to 10(-6) M GRF. These results demonstrate that NPY may directly modulate GH secretion at the level of the pituitary gland.  相似文献   

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