Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Receptors for gonadotropins and prostaglandins in lysosomes of bovine corpora lutea.

The total mitochondrial fraction of bovine corpus luteum specifically bound [³H]prostaglandin (PG) E₁, [³H] PGF_2α, and ¹²⁵I-labeled human lutropin (hLH) despite very little 5′-nucleotidase activity, a marker for plasma membranes. Since the total mitochondrial fraction isolated by conventional centrifugation techniques contains both mitochondria and lysosomes, it was subfractionated into mitochondria and lysosomes to ascertain the relative contribution of these fractions to the binding. Subfractionation resulted in an enrichment of cytochrome c oxidase (a marker for mitochondria) in mitochondria and of acid phosphatase (a marker for lysosomes) in lysosomes. The lysosomes exhibited little or no contamination with Golgi vesicles, rough endoplasmic reticulum, or peroxisomes as assessed by their appropriate marker enzymes. Subfractionation also re ulted in [³H] PGE₁, [³H] PGF_2α, and ¹²⁵I-labeled hLH binding enrichment with respect to homogenate in lysosomes but not in mitochondria. The lysosomal binding enrichment and recovery were, however, lower than in plasma membranes. The ratios of marker enzyme to binding, an index of organelle contamination, revealed that plasma membrane and lysosomal receptors were intrinsic to these organelles. Freezing and thawing had markedly increased lysosomal binding but had no effect on plasma membrane binding. Exposure to 0.05% Triton X-100 resulted in a greater loss of plasma membrane compared to lysosomal binding. In summary, the above results suggest that lysosomes, but not mitochondria, in addition to plasma membranes, intrinsically contain receptors for PGs and gonadotropins. Furthermore, lysosomes overall contain a greater number of PGs and gonadotropin receptors compared to plasma membranes and these receptors are associated with the membrane but not the contents of lysosomes.     

Cellular distribution and cycle phase dependency of gonadotropin and eicosanoid binding sites in bovine corpora lutea.

Bovine luteal functions are regulated by gonadotropins and eicosanoids. The specific binding sites that presumably mediate the actions of these regulatory agents have previously been characterized in bovine luteal tissue. However, the cellular distribution and/or the cycle phase dependency of these binding sites have never been investigated. In the present study, we investigated these parameters by using quantitative light microscope autoradiography. The results showed that both small and large luteal cells contained binding sites for LH/hCG, prostaglandin (PG)E2, PGF2 alpha, PGI2, and leukotriene (LT)C4. In addition, luteal blood vessels contained LH/hCG and LTC4 binding sites and luteal fibroblasts contained PGE2 binding sites. On a per cell basis, there were more binding sites for all ligands in large luteal cells as compared to small or nonluteal cells. After correction for the cellular area differences, small luteal cells contained more LH/hCG, PGE2, PGI2, and LTC4 binding sites, while large luteal cells contained more PGF2 alpha binding sites. The small and large luteal cell binding of hCG, PGE2, PGI2, and LTC4 increased from early to mid luteal phase, followed by a decline in the late luteal phase. PGF2 alpha binding, on the other hand, increased from early to late luteal phase. In contrast to luteal cells, binding of hCG and LTC4 to luteal blood vessels and binding of PGE2 to luteal fibroblasts did not change during the cycle. These results suggest that LH/hCG and eicosanoid regulation of luteal function is more complex than previously envisioned and it involves both small and large luteal cells and, in some cases, also nonluteal cells.     

Characterization of gonadotropin binding sites in the intracellular organelles of bovine corpora lutea and comparison with plasma membrane sites

The specific binding of 125I-human choriogonadotropin (hCG) to plasma membranes, nuclear membranes, lysosomes, rough endoplasmic reticulum, heavy golgi, and medium and light golgi of bovine corpora lutea was dependent on the amount of protein, 125I-hCG concentration and incubation time. The bound hormone in all the organelles was able to rebind to fresh corresponding organelles. Scatchard analysis revealed a homogenous population of gonadotropin binding sites in plasma membrane, rough endoplasmic reticulum, heavy golgi, and medium and light golgi, whose binding affinities (Kd = 8.6-11.0 X 10(-11) M) were similar but whose number of available gonadotropin binding sites varied. Scatchard analyses of nuclear membranes and lysosome binding, on the other hand, were heterogenous (Nuclear membranes, 11 and 23 X 10(-11) M lysosomes, 3.4 and 130 X 10(-11) M). The rate constants for association (5.9 to 12.1 X 10(6) M-1 S-1) and dissociation (7.4 to 9.0 X 10(-4) S-1) were similar among different subcellular organelles except for nuclear membranes and lysosomes, where rate constants for association were significantly lower. The ligand binding specificity, lower effectiveness of human luteinizing hormone as compared to hCG in competition, the optimal pH, the lack of ionic requirements for binding, and the molecular size of 125I-hCG-gonadotropin binding site complexes solubilized from various intracellular organelles were similar to those observed for plasma membranes. Numerous differences were also observed between intracellular organelles and plasma membranes as well as among intracellular organelles themselves with respect to binding losses due to exposure to low and high pH values, di- and monovalent cations, increasing preincubation temperatures, and a variety of enzymes and protein reagents. The possible reasons for these similarities as well as differences observed are discussed. The differences are viewed as an additional indication that contamination cannot solely explain the presence of gonadotropin binding sites in various intracellular organelles.     

Cytokeratin expression in bovine corpora lutea

Cytokeratin (CK)-positive cells were obtained from bovine corpora lutea. When cultured, these cells behave like CK-positive endothelial cells obtained from bovine large blood vessels. The origin of CK-positive cells has now been studied in 45 bovine corpora lutea of different estrous cycle stages. Additionally, 7 corpora lutea of pregnant cows were examined. The tissues were grouped into early stage (days 2 to 4), secretory stage (days 5 to 17) and late stage (days 18 to 21) according to gross morphology, wet weight and total progesterone content. One portion of a corpus luteum was used for immunohistochemistry, and another for Western blot analysis. Twenty-six of the 45 corpora lutea showed CK expression, as confirmed by immunostaining and Western blotting. Cytokeratin expression was found in all corporalutea from the early stage, in 14 of 26 corpora lutea from the secretory stage, and 3 of 10 from the late stage. Early stage corpora lutea displayed zonation such that a high number of CK-positive luteal cells occurred in the region of the previous granulosa layer and a very low number in the previous thecal layer. Secretory CK-positive corpora lutea showed uniformly distributed, predominantly large luteal cells. In secretory corpora lutea of group A, CK-positive cells and a distinct microvascular tree were seen, the latter visualized by factor VIII-related antigen immunolabelling of endothelial cells. Group B showed none or very few CK-positive cells. Corpora lutea of pregnant cows behaved like corpora lutea of group B. Roughly 1% of CK-positive cells closely associated with the capillary wall were sometimes reminiscent of endothelial cell sprouts.     

The presence of leukotriene C4-binding sites in bovine corpora lutea of pregnancy

The presence of binding sites for [3H]leukotriene (LT) C4 in bovine corpora lutea of pregnancy was investigated with quantitative light microscopic autoradiography. Silver grains were found over small (15-20 microns) and large (20-50 microns) luteal cells and arteriolar smooth muscle. Vascular endothelial cells, erythrocytes in arteriolar lumen, and fibroblasts, on the other hand, contained very few or no net grains. The grain distribution over luteal cells and arteriolar smooth muscle was reduced (p less than 0.001) after coincubation with excess unlabeled LTC4 but not with excess unlabeled LTA4, LTB4, LTD4, LTE4, prostaglandin (PG)E2, PGF2 alpha or PGI2. The large luteal cells contained 16.1 net grains per cell, which was 6.4 and 7.0 times the number of specific grains as in small luteal and arteriolar smooth muscle cells, respectively (p less than 0.001). When the net grains were corrected for cell area differences, large luteal cells and arteriole smooth muscle cells contained a similar number of grains-which was two times as many as those found in small luteal cells. These findings suggest that LTC4 can potentially regulate functions of not only luteal cells but also luteal vasculature.     

Quantitative echotexture analysis of bovine corpora lutea

A study was designed to evaluate the attributes of ultrasound images of bovine ovarian CL throughout the estrous cycle. The ovaries of 8 heifers were examined daily by transrectal ultrasonography for 2 interovulatory intervals (ovulation = Day 0). Ultrasonographic examinations of the ovaries were videotaped daily, and recorded images of the CL were digitized for computer analysis of echotexture (mean pixel value and heterogeneity). Blood samples were taken daily and to determine plasma progesterone concentrations. Corpora lutea were of 2 morphological types, those with a central fluid-filled cavity (n = 6) and those without (n = 9). No differences were detected between CL with or without a fluid-filled cavity; therefore, data were combined. Mean pixel values of ultrasound images of the CL changed (P = 0.0001) during the interovulatory interval; values decreased (P < 0.05) from Day 0 to Day 3 during early growth of the CL, reached a plateau when increases in luteal diameter ceased, and decreased (P < 0.05) to minimal levels at the onset of regression of the CL. The mean pixel value subsequently increased (P < 0.05) after Day 17 to values similar to those at the beginning of the interovulatory interval. A time-dependent effect was not observed for heterogeneity of images of the CL (P > 0.5). The results supported the hypothesis that quantitative changes in luteal echotexture are reflective of changes in the physiologic status of the CL.     

Oestrogen specific binding sites in bovine muscle nuclei

A method for preparation of purified bovine diaphragm nuclei was developed and the presence of specific oestradiol binding sites was demonstrated in salt extracts of such muscle nuclei by kinetic, equilibrium and competition studies. Scatchard analysis of [3H]zeranol binding also indicated the presence of high-affinity binding sites in nuclei from female animals (heifers) for this synthetic oestrogenic anabolic agent. Measured levels of specific [3H]oestradiol binding were higher in zeranol-treated steer than in untreated heifer, or steer diaphragm nuclei. A second, lower-affinity oestrogen-binding component was identified using [3H]oestradiol at concentrations greater than 8 nM in all three types of animals. The data suggest that gonadal oestrogens or related anabolic agents might have direct effects on muscle through receptor-like macromolecules.     

Progesterone synthesis and histology of postpartum bovine corpora lutea

In vitro progesterone (P(4)) synthesis by corpora lutea (CL) from the first, second or third ovulation after calving was compared and correlated with their histology and cytology. The CL were removed 7 to 12 days after ovulation, and luteal cells isolated by digestion with collagenase. The response of isolated cells to luteinizing hormone (LH) was determined. Hematoxylin-eosin stained tissues were used to study histology, and the distribution of cell types was estimated by stereological methods. Ovulation occurred within 25 days of calving and interovulatory intervals were short, 12.1 +/- 3.9 days and 12.6 +/- 4.8 days, respectively. The CL removed after first ovulation were smaller and contained fewer live cells than those obtained after subsequent ovulations. Stimulation by LH in vitro was independent of cycle number or day of cycle but was related to the histology of the tissue. The CL composed of large cells (> 24 mum) with vacuolated cytoplasm contained high amounts of P(4) but were not stimulated by LH. Conversely, CL composed of small and medium- sized cells (10 to 20 mum) and/or intact larger cells contained little P(4) but were stimulated by LH. These observations indicate that the response of postpartum CL to LH in vitro is dependent upon the structural integrity of the tissue at the time of removal. Furthermore, these observations suggest that the short life of CL during the postpartum period may not be due to the absence of luteotrophic support, but to the action of a luteolytic mechanism.     

Subcellular distribution of prostaglandin and gonadotrop in receptors in bovine corpora lutea.

The subcellular distribution of prostaglandin (PG) E₁, F₂α and gonadotropin receptors in bovine corpora lutea was critically examined by preparing various subcellular fractions, assaying for various marker enzymes to assess the purity and examining ³H-PGE₁, ³H-PGF₂α and ¹²⁵I-human lutropin (hLH) specific binding. The marker enzyme data suggested that subcellular fractions were relatively pure with little or no cross contamination. The binding of ³H-PGs and ¹²⁵I-hLH was markedly enriched in plasma membranes with respect to homogenate. The other subcellular fractions also exhibited binding despite very little or no detectable 5′-nucleotidase activity. If 5′-nucleotidase was assumed to lack sensitivity and reliability to detect minor contamination with plasma membranes and ³H-PGs or ¹²⁵I-hLH binding were used as sensitive plasma membrane markers, it was still difficult to explain binding in other fractions based on plasma membrane contamination. Therefore, these results lead to the inevitable conclusion that plasma membranes were primary (or one of the primary) but not exclusive sites for PGE₁, PGF₂α and gonadotropin receptors.     

Sensitivity of bovine corpora lutea to prostaglandin F2alpha is dependent on progesterone, oxytocin, and prostaglandins.

Prostaglandin (PG) F2alpha that is released from the uterus is essential for spontaneous luteolysis in cattle. Although PGF2alpha and its analogues are extensively used to synchronize the estrous cycle by inducing luteolysis, corpora lutea (CL) at the early stage of the estrous cycle are resistant to the luteolytic effect of PGF2alpha. We examined the sensitivity of bovine CL to PGF2alpha treatment in vitro and determined whether the changes in the response of CL to PGF2alpha are dependent on progesterone (P4), oxytocin (OT), and PGs produced locally. Bovine luteal cells from early (Days 4-5 of the estrous cycle) and mid-cycle CL (Days 8-12 of the estrous cycle) were preexposed for 12 h to a P4 antagonist (onapristone: OP; 10(-4) M), an OT antagonist (atosiban: AT; 10(-6) M), or indomethacin (INDO; 10(-4) M) before stimulation with PGF2alpha. Although OP reduced P4 secretion (p < 0.001) only in early CL, it reduced OT secretion in the cells of both phases examined (p < 0.001). OP also reduced PGF2alpha and PGE2 secretion (p < 0.01) from early CL. However, it stimulated PGF2alpha secretion in mid-cycle luteal cells (p < 0.001). AT reduced P4 secretion in early and mid-cycle CL (p < 0.05). Moreover, PGF2alpha secretion was inhibited (p < 0.05) by AT in early CL. The OT secretion and the intracellular level of free Ca2+ ([Ca2+]i) were measured as indicators of CL sensitivity to PGF2alpha. PGF2alpha had no influence on OT secretion, although [Ca2+]i increased (p < 0.05) in the early CL. However, the effect of PGF2alpha was augmented (p < 0.01) in cells after pretreatment with OP, AT, and INDO in comparison with the controls. In mid-cycle luteal cells, PGF2alpha induced 2-fold increases in OT secretion and [Ca2+]i. However, in contrast to results in early CL, these increases were magnified only by preexposure of the cells to AT (p < 0.05). These results indicate that luteal P4, OT, and PGs are components of an autocrine/paracrine positive feedback cascade in bovine early to mid-cycle CL and may be responsible for the resistance of the early bovine CL to the exogenous PGF2alpha action.     

Comparisons of gonadotropin binding sites and progesterone concentrations among the corpora lutea of individual pigs

In polyovular species, it is unclear whether the characteristics of each individual corpus luteum (CL), such as mass, progesterone concentration and receptors for luteinizing hormone (LH), are representative of those of its cohorts during the ovarian cycle. The current study was performed 1) to characterize the conditions for estimation of binding parameters for LH receptors in porcine CL, and 2) to compare LH binding sites, luteal progesterone concentrations and luteal masses among CL of ovaries within individual pigs. Gonadotropin binding sites in porcine CL were characterized via specific binding of 125I-human (h) LH to 20,000 X g particulate fractions of luteal tissue. Specific binding was directly proportional to tissue content and was detectable at the lowest content tested (0.5 mg tissue equivalents/tube). Specific uptake of 0.25 ng LH by 5.0 mg tissue equivalents was time- and temperature-dependent; steady-state binding was achieved within 20 h at 37 and 25 degrees C. Binding of LH after 20 h incubation at 37 degrees C (4718 +/- 192 cpm, means +/- SEM) and 25 degrees C (4112 +/- 340 cpm) was greater than that at 4 degrees C (1930 +/- 5 cpm, P less than 0.01). Luteal particulates from individual CL of ovaries collected from four mature nonpregnant pigs (13-23 CL/pig) were incubated with eight concentrations of 125I-hLH. Steady-state binding depended upon hormone concentration until reaching saturation at 2.5 ng 125I-hLH/tube. Scatchard analyses yielded linear plots. Binding capacities for LH ranged among pigs from 0.71 +/- 0.03 to 3.69 +/- 0.13 fmol/mg CL equivalents and receptor affinities (Kd) ranged from 0.92 +/- 0.05 to 4.89 +/- 0.41 X 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of prostaglandins and progesterone by cells from corpora lutea of mares

Corpora lutea (CL) were collected from mares during early (Day 4-5), mid- (Day 8-9), and late (Day 12-13) dioestrus. Dispersed cell suspensions were obtained by enzymic digestion of tissue. Two distinct luteal cell populations (large and small) were observed. The proportion of small luteal cells significantly increased as age of CL advanced. Cells (2 x 10(6)) from CL which were incubated for 24 h secreted prostaglandin (PG) F, PGE-2 and 6-keto-PGF-1 alpha (the stable metabolite of prostacyclin). Higher concentrations of all PGs were produced by cells from CL at early dioestrus than from those at mid- or late dioestrus. The ratio of PGF:PGE-2 increased from 0.33 in CL of early dioestrus to 1.34 in CL of mid-dioestrus, whereas ratios of PGF:6-keto-PGF-1 alpha remained relatively constant (approximately 0.6). The ratio of PGE-2:6-keto-PGF-1 alpha from CL decreased between early (3.27) and mid-dioestrus (0.43). Addition of LH, dbcAMP, or ionophore to cell cultures did not consistently affect secretion of progesterone or PGs by luteal cells. It is suggested that prostaglandins produced by luteal cells of mares may contribute to control of luteal function and that the changing ratios of prostaglandins may be more important in controlling the lifespan of the CL than absolute concentrations of each.     

Prostaglandin E1 and F2alpha specific binding in bovine corpora lutea: comparison with luteolytic effects.

Preliminary characterization indicated the presence of separate prostaglandin (PG)E1 and (PG)F2alpha binding sites in membrane fractions prepared from bovine corpora lutea. These differ in the rate and temperature dependence of the specific binding. Equilibrium binding data indicate the apparent dissociation constants as 1.32 x 10(-9)M and 1.1 x 10(-8)M for PGE1 and PGF2alpha, respectively. Competition of several natural prostaglandins for the PGE1 and PGF2alpha bovine luteal specific binding sites indicates specificity for the 9-keto or 9alpha-hydroxyl moiety, respectively. Differences in relative ability to inhibit 3H-PG binding were found due to sensitivity to the absence or presence of the 5, 6-cis-double bond as well. Bovine luteal function was affected following treatment of heifers with 25 mg PGF2alpha as measured by reduced estrous cycle length, decreased corpus luteum size and significantly decreased plasma progesterone levels. In contract, treatment with 25 mg PGE1 resulted in cycle lengths comparable to those of non-treated herdmates with no apparent modification in corpus luteum size. However, plasma progesterone levels were increased significantly following PGE1 treatment compared to pretreatment values. In so far as data obtained in vitro on PGF2alpha relative binding affinity to the bovine CL can be compared to data obtained independently in vitro on PGF2alpha induced luteolysis in the bovine, PGF2alpha relative binding to the CL and luteolysis appeared to be associated. By similar reasoning, there was no apparent relationship between PGE1 relative binding affinity in the luteal fractions and luteolysis in estrous cyclic cattle.     

Prostaglandin F2alpha specific binding in equine corpora lutea

Preliminary studies indicate the presence of PGF2alpha specific binding sites in membrane fractions prepared from equine corpora lutea. The equilibrium binding data indicate an apparent dissociation constant of 3.2 X 10(-9)M and the concentration of binding sites of -0.1 pmoles/mg membrane protein. Competition of several natural prostaglandins for equine luteal PGF2alpha specific binding sites indicates specificity for the 9alpha-hydroxyl moiety and the 5,6-cis doublebond. Significant increases in relative binding affinities were demonstrated for PGF2alpha analogs with a phenyl ring introduced at carbons 16 or 17. Specific PGF2alpha binding was demonstrated in corpora lutea collected at known stages of the estrous cycle. There was no pattern in these values based on the stage of the cycle. While specific 3H-PGE1 binding could be demonstrated, no high affinity sites could be quantitated. 3H-PGE1 binding appeared unaffected by changes in temperature or time of incubation, whereas PGF2alpha specific binding was significantly modified by both these factors.     

Occurrence and properties of a prostaglandin F2alpha receptor in bovine corpora lutea.

Prostaglandin F2alpha was specifically bound by a particulate fraction from bovine corpora lutea. The rate constants for the association (7.5 X 10(3) M-1 S-1) and dissociation (2.1 X 10-4 S-1) reactions gave a dissociation constant of 2.8 X 10(-8) M which is similar to that determined from a Scatchard plot of binding data at equilibrium (5 X 10(-8) M). The receptor was stable for several hours at 23 degrees C but was rapidly destroyed at 37 degrees C. The pH optimum for the binding reaction was 6.3. The receptor had high specificity for prostaglandin F2alpha and had much lower affinities for other prostaglandins. Luteinizing and follicle-stimulating hormones had no effect on the prostaglandin F2alpha-receptor interaction.